INVESTIGATIONS IN FISH CONTROL

63. Laboratory Efficacy of

3-Trifluoromethyl-4-nitrophenol 

(TFM) as a Lampricide

64. Effects of 3-Trifluoromethyl-4-nitrophenol

(TFM) on Developmental Stages of the Sea Lamprey

65. Accumulation and Loss of Residues of 

3-Trifluoromethyl-4-nitrophenol (TFM) 

in Fish Muscle Tissue: Laboratory Studies

66. Residues of 3-Trifluoromethyl-4-nitrophenol 

(TFM) in a Stream Ecosystem after Treatment 

for Control of Sea Lampreys

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.
INVESTIGATIONS IN FISH CONTROL

63. Laboratory Efficacy of 3-Trifluoromethyl-4- 

nitrophenol (TFM) as a Lampricide

By Verdel K. Dawson, Kenneth B. Gumming, 

and Philip A. Gilderhus

64. Effects of 3-Trifluoromethyl-4-nitrophenol (TFM) 

on Developmental Stages of the Sea Lamprey

By George V. Piavis and John H. Howell

65. Accumulation and Loss of Residues of 

3-Trifluoromethyl-4-nitrophenol (TFM) 

in Fish Muscle Tissue: Laboratory Studies

By Joe B. Sills and John L. Alien

66. Residues of 3-Trifluoromethyl-4-nitrophenol 

(TFM) in a Stream Ecosystem after Treatment 

for Control of Sea Lampreys

By Philip A. Gilderhus, Joe B. Sills, and 

John L. Alien

United States Department of the Interior

Fish and Wildlife Service 

Washington, B.C. 1975
IN THIS COVER

Pages

Laboratory Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM) as a 

Lampricide, by Verdel K. Dawson, Kenneth B. Gumming, and Philip A. 

Gilderhus........................................................... 1-13

Effects of 3-Trifluoromethyl-4-nitrophenol (TFM) on Developmental

Stages of the Sea Lamprey, by George V. Piavis and John H. Howell. 1- 8

Accumulation and Loss of Residues of 3-Trifluoromethyl-4-nitrophenol 

(TFM) in Fish Muscle Tissue: Laboratory Studies, by Joe B. Sills and 

John L. Alien ....................................................... 1-10

Residues of 3-Trifluoromethyl-4-nitrophenol (TFM) in a Stream Ecosystem 

after Treatment for Control of Sea Lampreys, by Philip A. Gilderhus, Joe 

B. Sills, and John L. Alien .......................................... 1-7
FOREWORD

Any program to develop data regarding the safe and efficacious use of a chemical 

must include investigations of its effects on nontarget organisms, on the target 

organism and its life stages, and should consider residue levels which might occur in 

natural ecosystems following application.

The lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), has been used extensively to control larvae of the sea lamprey, Petromyzon marinus, in the Great Lakes. 

Previous publications in Investigations in Fish Control (Nos. 56-62) have reported 

effects of TFM on algae, zooplankters, amphipods, isopods, crayfish, insects, and 

fishes under laboratory conditions. The following reports describe research on the 

effects of TFM on selected developmental stages of larval lampreys, and discuss 

residue levels that occur in fish tissue and in a stream ecosystem following treatment 

with TFM.

These papers represent part of a continuing series in Investigations in Fish 

Control which describes ecological effects of the use of TFM as a lampricide. The 

completed series of reports will be used to support petitions for the registration of 

TFM as an effective control for the sea lamprey.

Fred P. Meyer, Director 

Fish Control Laboratories

INVESTIGATIONS IN FISH CONTROL

63. Laboratory Efficacy of

3-Trifluoromethyl-4-nitrophenol 

(TFM) as a Lampricide

By Verdel K. Dawson, Kenneth B. Cumming, and Philip A. Gilderhus

United States Department of the Interiorj

Fish and Wildlife Service 

Washington, D.C. 1975
CONTENTS

Page

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

Introduction ............................................................ 4

Materials and methods.................................................. 4

Results ................................................................. 5

Species and sizes of lampreys ....................................... 6

Effect of temperature................................................ 6

Effect of water hardness ............................................ 6

Effect of pH ........................................................ 6

Effect of substrate .................................................. 6

Discussion.............................................................. 9

Conclusions............................................................. 10

Literature cited ......................................................... 10

Appendix ............................................................... 11
LABORATORY EFFICACY OF

3-TRIFLUOROMETHYL-4-NITROPHENOL(TFM)

AS A LAMPRICIDE

by

Verdel K. Dawson, Kenneth B. Gumming, and Philip A. Gilderhus 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT

The lampricidal activity of 3-trifluoromethyl-4-nitrophenol (TFM) was tested 

under controlled laboratory conditions to evaluate factors which may influence the 

efficacy of the chemical. TFM was tested at temperatures of 7,12,17, and 22 C, total 

water hardnesses of 12, 44,170, and 300 mg/1 as CaCOa, and pH's of 6.5,7.5,8.5, and 

9.0. TFM is an effective lampricide. It is more effective against larvae of the sea 

lamprey (Petromyzon marinus) than against embryos and prolarval stages and 

slightly more effective against larvae of sea lampreys than against those of the 

American brook lamprey (Lampetra lamottei). Efficacy of the lampricide is affected 

very little by temperature, but is reduced in hard water, especially at high pH's. High 

pH decreases the activity of TFM and has the greatest influence on toxicity of any of 

the factors investigated. TFM is significantly more effective against exposed (freeswimming) larvae than against those in burrows.

INTRODUCTION

In 1964, 3-trifluoromethyl-4-nitrophenol 

(TFM) was registered by the Pesticide Registration Division of the U.S. Department of 

Agriculture for limited use in tributaries of the 

Great Lakes to control the sea lamprey 

(Petromyzon marinus), against which the 

chemical had been shown to be selective 

(Applegate et al. 1958). In 1970, however, the 

registration was threatened with cancellation 

by the Environmental Protection Agency, and 

since then, extensions have been granted to 

provide time for preparation of information 

necessary to support continued registration.

A literature review by Schnick (1972) indicated that numerous tests of the lampricidal 

activity of TFM have been conducted in waters 

of various qualities and temperatures. However, 

because most of the early studies were conducted in natural waters from various sources 

and with different characteristics, the influence

of individual factors was difficult to evaluate 

(Applegate et al. 1961; Applegate and King 1962; 

Kanayama 1963; Smith 1966; Zimmerman 1968; 

and Johnson 1970).

Recent studies have defined the influence of 

water chemistry on the toxicity of TFM to 

nontarget aquatic organisms (Marking and 

Olson 1975; Kawatski et al. 1975; and Chandler 

and Marking 1975). Additional information is 

needed, however, on the efficacy of TFM as a 

lampricide. The purpose of the present study 

was to determine: the toxicity of TFM to 

different species, sizes, and developmental 

stages of lampreys; the individual and combined influences of temperature, water 

hardness, and pH on the toxicity of TFM; and 

the toxicity to burrowed and exposed (freeswimming) lamprey larvae at the same stage of 

development.
MATERIALS AND METHODS

We used electrofishing gear to collect lamprey 

larvae from the Rifle River watershed in eastern 

Michigan. The lampreys were anesthetized in 

100-mg/l solutions of MS-222 and sorted by 

species and size. The average lengths (and 

range in lengths) were 7 (4-9) cm for sea lamprey 

larvae and 7 (5-9) cm, and 16 (13-19) cm for two 

groups of American brook lamprey (Lampetra 

lamottei) larvae. The test organisms were 

placed in troughs containing a sifted sand 

substrate about 10 cm deep, and flowing well 

water at 12 C. The lampreys were maintained in 

the troughs for at least 2 wk prior to testing.

A three-dimensional testing model was used 

to evaluate the influences of temperature, 

hardness, and pH. Each of these factors was 

varied individually in the presence of selected 

combinations of the other two factors, so that 

the effect of each variable could be isolated and 

examined under a variety of controlled conditions.

The test water was deionized and 

reconstituted to four different hardnesses (Table 

1), and the pH was adjusted with appropriate 

buffers (Table 2). The solutions were checked

Table 1. Ingredients and characteristics of reconstituted waters used in toxicity tests

Water 

type

Very soft

Soft 0

Hard

Very hard

Salts added (mg/l)

NaHCO3

12

48

192

384

CaSO4 2H 20

7.5

30

120

240

MgSO4

7.5

30

120

240

KCI

0.5

2.0

8.0

16

pH 

range

6.4-6.8

7.2-7.6

7.6-8.0

8.0-8.4

Total 

hardness 

(mg/l CaCO3 )

10-13

40-48

160-180

280-320

Total 

alkalinity 

(mg/l CaCO 3 )

10-13

30-35

110-120

225-245

Standard reconstituted water used in routine toxicity tests.

Table 2. Quantities of buffering chemicals used for adjusting the pH 

in reconstituted water of various hardnesses

PH

6.5

7.5

8.5

9.0

Buffer

IN NaOH

1M KH 2PO 4

IN NaOH

1M KH 2 PO 4

IN NaOH

0.5M H 3 BO 3

IN NaOH

0.5M H 3 BO 3

Very soft

4

20

14

20

3.5

20

2

20

Ml of solutions per

Soft

5

30

14

20

6.5

40

5.5

20

15 liters of water

Hard

1

40

13

20

6

30

8

20

Very hard

_

60

12

20

10

30

11

20
daily with a pH meter and readjusted as 

necessary to maintain the desired pH. Water 

temperatures were controlled by placing the test 

vessels in water baths maintained at 7,12,17, or 

22 C.

Field grade TFM (35.7%, obtained from 

American Hoechst Corporation), which is 

formulated with N,N-dimethylformamide 

(DMF), was measured volumetrically and dissolved in water. Since the entire formulation is 

applied in streams, concentrations are reported 

on the basis of total formulated lampricide and 

expressed as /LI 1/1.

TFM was added to the test vessels about 20 h 

after the introduction of lampreys. We exposed 

10 lamprey larvae to each concentration of the 

chemical in 15-liter glass jars using methods 

similar to those described for static toxicity tests 

(Lennon and Walker 1964). Dead fish were 

counted and removed at 1, 3, 6, and 12 h after 

initial exposure, and daily thereafter, during the 

96-h tests. LCSO's, LC99's, and 95% confidence 

intervals were computed according to the 

methods of Litchfield and Wilcoxon (1949). A P 

value of 0.05 was used to evaluate significance.

The toxicity of TFM to sea lampreys at 

selected stages of development as defined by 

Piavis (1961) was determined in soft, 

reconstituted water at 17 C. We obtained test 

specimens by collecting sexually mature sea 

lampreys from nest areas, spawning them 

artificially, and incubating the eggs in the 

laboratory. The immature specimens were 

exposed to TFM when they reached the desired 

stages of development. In additional tests we 

exposed sea lamprey prolarvae (Stage 17, 

burrowing) in water of selected hardnesses and 

pH's at 17 C.

Since stage 18 larvae in nature usually live in 

burrows in the substrate, the difference in 

toxicity of TFM to burrowed and free-swimming 

lampreys at this stage of development was 

determined. To eliminate the effect of adsorption of the chemical by the substrate, we 

conducted these tests in a flow-through test 

apparatus similar to that used by Marking et al. 

(1975), in which the test solution was introduced 

continuously. Thus, the concentration of TFM 

was essentially identical in tests containing the 

burrowed and free-swimming larvae.

RESULTS

Species and Sizes of Lampreys

The 96-h LCSO's for TFM in soft, 

reconstituted water at 12 C for 7-cm sea 

lampreys and 7-cm American brook lampreys 

were 1.57 and 3.30 /xl/1 of formulated TFM, 

respectively representing a significant 

difference in toxicity (Table 3). The corresponding LC50 for 16-cm American brook lampreys 

was 2.41 /i 1/1, which was not significantly 

different from the values for smaller sizes of the 

two species.

The toxicity of TFM to five stages of sea 

lampreys increased as the development of the

Table 3. Toxicity of TFM (35.7%) to two species and sizes of 

lamprey larvae in soft, reconstituted water at 12 C

Species

Sea lamprey

American

brook lamprey

American

brook lamprey

Average 

length

(cm)

7

7

16

LC50 and

24 h

1.57 

1.10-2.06

4.45

2.92-6.77

2.52

1.95-3.25

95% confidence interval

48 h

1.57 

1.10-2.06

3.30

2.45-4.44

2.41

2.02-2.87

(All/1) at

96 h

1.57 

1.10-2.06

3.30

2.45-4.44

2.41

2.02-2.87
6

lampreys advanced (Table 4). This trend was 

evident at all exposure periods, and is exemplified by a decrease in the 48- and 96-h 

LCSO's from 8.65 to 1.57 M l/l of TFM as 

development progressed from Stage 14 

(hatching) to Stage 18 (7-cm larvae).

Effect of Temperature

There was little difference in the toxicity of 

TFM to sea lamprey larvae at temperatures of 7, 

12, 17, or 22 C (Table 5), regardless of the 

exposure period or the hardness or pH of the test 

water.

Table 4. Toxicity of TFM (35.7%) to various developmental stages of 

sea lamprey larvae in soft, reconstituted water at 17 C

Stage"

14 (hatching)

15 (pigmentation)

16 (gill cleft)

17 (burrowing)

18 (7-cm larvae)

LC50 and 95%

24 h

10.0 

8.24-12.1

6.20 

4.67-8.23

6.20 

5.37-7.16

3.88 

3.14-4.84

1.57 

1.10-2.06

confidence interval (fJL\/

48 h

8.65 

7.83-9.55

6.20 

4.67-8.23

5.60 

5.07-6.18

3.88 

3.14-4.84

1.57 

1.10-2.06

'!) at

96 h

8.65 

7.83-9.55

6.15 

4.65-8.14

5.60 

5.07-6.18

3.88 

3.14-4.84

1.57 

1.10-2.06

"According to Piavis (1961).

Effect of Water Hardness

The toxicity of formulated TFM to sea 

lamprey larvae declined as water hardness 

increased, especially at the higher pH's. For 

example, the 12-h LC99's at 12 C and 12 and 300 

mg/1 of total hardness, were 0.950 and 1.58 ul/1 

of TFM, respectively, at pH 6.5 and 8.50 and 32.5 

M1/1 at pH 8.5 (Table 5).

Similar effects of water hardness on the 

toxicity of TFM to 7-cm sea lamprey larvae also 

were evident for mortalities reported as LC50's 

and at all exposure periods (See Appendix). The 

influence of water hardness on the toxicity of 

TFM was similar in tests against sea lamprey 

prolarvae at Stage 17 (Table 6).

Effect of pH

The pH of the test water had more influence on 

the toxicity of TFM to sea lampreys than either 

temperature or hardness. In soft water at 12 C 

the 12-h LC99's for 7-cm larvae at pH 6.5, 7.5, 

8.5, and 9.0 were 1.17, 4.10,12.0, and 33.0 n\/\ of

TFM, respectively (Table 5). The influence of pH 

on the activity of TFM was observed at all 

temperatures and water hardnesses tested, but 

was especially evident in the harder waters. 

This general toxicity pattern also applied to 

Stage 17 prolarvae (Table 6).

Effect of Substrate

Sea lampreys burrowed in sand are less 

vulnerable to TFM than are lampreys confined 

without a substrate (Table 7). A concentration of 

5.2 /ul/1 killed 100% of the free-swimming 

lampreys in 4 h, but did not kill all of the 

burrowed lampreys until after 48 h. At 24 h, the 

LC50 for burrowed lampreys (4.75 M!/!) was 

more than four times that for free-swimming 

lampreys (1.17 /xl/1). A concentration of 1.7 /xl/1 

produced 40% mortality among the freeswimming lampreys in 12 h and 100% mortality 

in 48 h, but this concentration failed to kill any 

of the burrowed lampreys in 96 h.
Table 5. Toxicity of TFM (35.7%) to 7-cm sea lamprey larvae after 12 h of 

exposure in waters of selected temperatures, pH's, and hardnesses

Water 

pH hardness 

(mg/l CaC0 3 )

6.5 12

44

170

300

7.5 12

44

170

300

8.5 12

44

170

300

9.0 12

44

170

12-h LC99 and 95% confidence 

interval (/ill/I) at temperatures (°C) of

7

0.860 

0.682-1.08

1.24 

0.790-1.95

1.00 

0.699-1.43

1.60 

1.28-2.00

2.32 

1.92-2.81

2.61 

1.66-4.10

5.10 

3.86-6.73

10.0 

6.99-14.3

11.0 

8.33-14.5

29.5 

24.4-35.7

27.6 

22.8-33.4

35.0 

28.0-43.8

...

_

51.5 

39.0-68.0

12

0.950 

0.785-1.15

1.17 

0.886-1.54

1.02 

0.843-1.23

1.58 

1.01-2.48

2.90 

2.03-4.15

4.10 

2.87-5.86

6.80 

4.76-9.72

6.61 

5.28-8.26

8.50 

5.41-13.3

12.0 

8.39-17.2

18.2 

14.6-22.8

32.5 

24.6-42.9

8.40 

6.36-11.1

33.0 

23.1-47.2

41.0 

28.7-58.6

17

1.00 

0.800-1.25

1.25 

1.00-1.56

1.10 

0.880-1.38

1.62 

1.13-2.32

2.73 

2.07-3.60

4.15 

3.32-5.19

5.18 

4.28-6.27

6.40 

5.29-7.74

10.0 

7.58-13.2

21.1 

16.9-26.4

33.0 

23.1-47.2

27.5 

17.5-43.2

..-

58.0 

47.9-70.2

22

1.30 

0.985-1.72

1.25 

0.874-1.79

2.00 

1.52-2.64

1.63 

1.30-2.04

3.50 

2.80-4.38

3.80 

3.14-4.60

6.50 

4.92-8.58

7.03 

5.81-8.51

12.5 

10.0-15.6

22.1 

16.7-29.2

23.7 

16.6-33.9

29.6 

23.7-37.0

66.2 

54.7-80.1
Table 6. Toxicity of TFM (35.7%) to sea lamprey prolarvae (Stage 17) 

in waters of selected hardness and pH at 17 C

Water 

pH hardness 

(mg/l CaC03 )

6.5 12

44

170

300

7.5 12

44

170

300

8.5 12

44

170

300

9.0 12

44

170

300

LC50 and 95% confidence interval (/Ill/I) at

3 h

1.40 

1.13-1.73

1.40 

1.11-1.76

1.74 

1.49-2.04

1.41 

1.14-1.74

6.40 

5.12-7.99

6.75 

5.64-8.08

5.65 

4.57-6.99

7.99 

6.55-9.75

34.5 

29.6-40.3

>40.0°

74.0 

57.4-95.5

60.0 

54.1-66.5

62.0 

55.2-69.7

62.1 

51.3-75.2

>100 a

83.0 

71.8-96.0

6 h

1.40 

1.13-1.73

1.40 

1.11-1.76

1.74 

1.49-2.04

1.41 

1.14-1.74

6.35 

5.06-7.97

4.40 

3.69-5.25

5.65 

4.57-6.99

7.99 

6.55-9.75

22.0 

19.6-24.7

32.0 

26.4-38.8

40.5 

33.8-48.5

46.0 

40.8-51.9

61.9 

54.7-70.1

54.6 

46.0-64.9

71.5 

61.4-83.3

81.6 

64.4-103

12 h

0.710 

0.531-0.949

1.21 

0.943-1.55

1.74 

1.49-2.04

1.41 

1.14-1.74

3.64 

2.93-4.52

3.90 

3.34-4.56

5.65 

4.57-6.99

6.25 

5.48-7.12

15.0 

13.0-17.3

32.0 

26.4-38.8

40.0 

33.0-48.6

31.5 

31.1-31.9

46.0 

40.8-51.9

46.0 

42.5-49.8

62.0 

54.4-70.7

37.1 

29.6-46.4

24 h

0.610 

0.492-0.757

1.21 

0.943-1.55

1.74 

1.49-2.04

1.41 

1.14-1.74

3.49 

2.94-4.14

3.88 

3.14-4.84

5.65 

4.57-6.99

5.20 

4.31-6.27

10.1 

7.84-13.0

27.5 

25.6-29.5

35.1 

29.8-41.3

24.0 

20.7-27.9

29.3 

26.4-32.6

26.0 

18.7-36.1

55.5 

46.5-66.3

35.5 

28.9-43.7

48 h

0.610 

0.492-0.757

1.21 

0.943-1.55

1.23 

0.955-1.58

1.41 

1.14-1.74

3.49 

2.94-4.14

3.88 

3.14-4.84

5.65 

4.57-6.99

5.20 

4.31-6.27

10.0 

8.00-12.5

17.3 

15.5-19.3

35.1 

29.8-41.3

24.0 

20.7-27.9

29.3 

26.4-32.6

26.0 

18.7-36.1

45.0 

39.5-51.2

30.5 

23.7-39.2

96 h

0.610 

0.492-0.757

1.21 

0.943-1.55

1.23 

0.955-1.58

1.41 

1.14-1.74

3.49 

2.94-4.14

3.88 

3.14-4.84

5.02 

4.11-6.13

5.20 

4.31-6.27

9.50 

7.55-12.0

17.2 

14.4-20.6

35.1 

29.8-41.3

24.0 

20.7-27.9

24.5 

21.1-28.4

22.0 

16.9-28.7

43.5 

35.8-52.9

26.0 

18.7-36.1

No mortality at highest concentration tested.
DISCUSSION

Piavis (1962) recognized that the sensitivity of 

sea lamprey to TFM increased in the more 

advanced stages of embryonic development, 

and suggested that control of the sea lamprey 

would be most effective if conducted at a time 

when the lampreys reached the more sensitive 

larval stage. Information on the toxicity of TFM 

to sea lampreys of various developmental stages 

presented in this paper supports his findings.

Table 7. Toxicity of TFM (35.7%) to burrowed 

and free-swimming sea lamprey larvae in 

a flow-through diluter containing carbonfiltered city water at 12 C

Exposure LC50 and 95% confidence interval ( fJi\/\) for

time 

(hours)

3

4

6

7

12

24

48

96

Free-swimming 

lampreys

6.50

5.19-8.13

4.61

4.25-5.00

4.06

3.73-4.43

3.17

2.64-3.81

1.71

1.55-1.88

1.17

1.02-1.35

<1.07

<1.07

Burrowed 

lampreys

>7.00

>7.00

>7.00

6.85

5.42-8.66

5.20

_ a

4.75

3.80-5.93

2.99

2.57-3.48

2.50

2.22-2.81

'Data insufficient to compute confidence intervals.

The reduced toxicity of TFM at the higher 

pH's presumably results from an increased 

ionization of the molecule (pKa = 6.07; Applegate 

et al. 1961). The un-ionized form of certain 

molecules is lipid-soluble, and therefore more 

easily transported across the gills of fish (Sills 

and Alien 1971).

The reduced toxicity of TFM in hard water, 

especially at the higher pH's, may result from 

the formation of a complex between the ionized 

form of the TFM molecule and divalent cations. 

As the hardness of the water increases, the 

availability of more cations for complexing 

shifts the ionization equilibrium and results in a 

decrease in the concentration of the more active, 

un-ionized form of TFM.

Although temperature changes have been 

blamed for incomplete kills during stream 

treatments (U.S. Bureau of Commercial 

Fisheries 1958; Smith and King 1970), 

laboratory studies have indicated that 

temperature has little effect on the toxicity of 

TFM (U.S. Bureau of Commercial Fisheries 

1960; Applegate et al. 1961). However, 

Applegate et al. (1961) reported that the rate of 

death slowed as the temperature decreased and 

that the selectivity against lampreys increased 

as the temperature dropped near freezing.

We found that free-swimming lampreys were 

more vulnerable to TFM than burrowed lampreys. Presumably the free-swimming lampreys 

are more excited and their rate of metabolism 

and uptake is greater than that of the burrowed 

lampreys. Also, the burrowed lampreys may be 

somewhat protected from exposure to TFM in 

the water. Since the field-use concentration of 

TFM for each stream has been determined by 

on-site bioassays of TFM against freeswimming lampreys, these tests could indicate 

treatment concentrations which are insufficient 

to produce complete elimination of the burrowed 

lamprey unless the lethal concentrations are 

maintained over an extended period. These 

results do not support those of Applegate et al. 

(1958) who reported that concentrations of TFM 

lethal to all larval lampreys were essentially the 

same in jar tests and in treatments of a 

simulated stream.
10

CONCLUSIONS

1. TFM is effective as a lampricide.

2. Sea lampreys are more sensitive to TFM than 

American brook lampreys, but the difference 

is not enough to permit selective removal of 

sea lampreys.

3. Early developmental stages of sea lampreys 

are more resistant to TFM than the larval 

stage.

4. Temperature has very little influence on the 

toxicity of TFM.

5. The toxicity of TFM is significantly reduced 

in water of high pH.

6. The lampricide is less toxic in hard than in 

soft water, especially at high pH's.

7. Burrowed sea lamprey larvae are significantly less vulnerable to TFM than are freeswimming sea lamprey larvae.

REFERENCES

Applegate, V.C., J. H. Howell, J. W. Moffett, B. G. H. 

Johnson, and M. A. Smith. 1961. Use of 3-trifluormethyl-4-nitrophenol as a selective sea lamprey larvicide. 

Great Lakes Fish. Comm., Tech. Rep. No. 1. 35 pp.

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

mononitrophenols containing halogens as selective sea 

lamprey larvicides. Science 127(3294):336-338.

Applegate, V. C., and E. L. King, Jr. 1962. Comparative 

toxicity of 3-trifluormethyl-4-nitrophenol (TFM) to 

larval lampreys and eleven species of fishes. Trans. Am. 

Fish. Soc. 91(4):342-345.

Chandler, J. H., and L. L. Marking. 1975. Toxicity of the 

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

selected aquatic invertebrates and frog larvae. U.S. Fish 

Wildl. Serv., Invest. Fish Control No. 62. 7 pp.

Johnson, B. G. H. 1970. Water quality and biological activity 

of lampricide, Appendix 20. Pages 90-93 in Great Lakes 

Fishery Commission, Report of annual meeting, St. Paul, 

Minn., June 16-18, 1970.

Kanayama, R. K. 1963. The use of alkalinity and conductivity measurements to estimate concentrations of 3-trifluormethyl-4-nitrophenol required for treating lamprey 

streams. Great Lakes Fish. Comm., Tech. Rep. No. 7. 10 

pp.

Kawatski, J. A., M. M. Ledvina, and C. R. Hansen. 1975. 

Actue toxicities of 3-trifluoromethyl-4-nitrophenol 

(TFM) and 2',5-dichloro-4'-nitrosalicylanilide (Bayer 73) 

to larvae of the midge Chironomus tentans. U.S. Fish 

Wildl. Serv., Invest. Fish Control, No. 57. 7 pp.

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

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

Wildl. Serv., Invest. Fish Control No. 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. 99(2):99-113.

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

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

U.S. Fish Wildl. Serv., Invest. Fish Control No. 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 No. 60. 13 pp.

Piavis, G. W. 1961. Embryological stages in thesealamprey 

and effects of temperature on development. U.S. Fish 

Wildl. Serv., Fish. Bull. 61:111-143.

Piavis, G. W. 1962. Exposure of several developmental 

stages of the sea lamprey, Petromyzon marinus, to 

selective larvicides. Copeia 1962 (3):652-653.

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

(3-trifluromethyl-4-nitrophenol) as a lampricide. U.S. 

Fish Wildl. Serv., Invest. Fish Control No. 44. 31 pp.

Sills, J. B., and J. L. Alien. 1971. The influence of pH on the 

efficacy and residues of quinaldine. Trans. Am. Fish. Soc. 

100 (3):544-545.

Smith, B. R. 1966. Lamprey control and research in the 

United States. Pages 31-45 in Great Lakes Fishery 

Commission annual report for the year 1964.

Smith, B. R., and E. L. King, Jr. 1970. Lamprey control in the 

United States. Pages 29-40 in Great Lakes Fishery 

Commission annual report for the year 1969.

U.S. Bureau of Commercial Fisheries. 1958. Lamprey 

control and research in the United States. Pages 34-50 in 

Great Lakes Fishery Commission annual report for the 

year 1958.

U.S. Bureau of Commercial Fisheries. 1960. Sea lamprey 

control and research in the United States in 1960, 

Appendix IV. Pages 1-9 in Great Lakes Fishery Commission, Report of annual meeting, Cleveland, Ohio, Dec. 1-2, 

1960.

Zimmerman, J. W. 1968. Water quality of streams tributary 

to Lakes Superior and Michigan. U.S. Fish Wildl. Serv., 

Spec. Sci. Rep. Fish. No. 559. 41 pp.
11

Appendix. Toxicity of TFM (35.7%) to free-swimming 7-cm sea lamprey larvae 

in waters of selected temperatures, pH's, and hardnesses

Total 

, 0 Jr pH hardness 

( } (mg/l CaCO 3 )

7 6.5 12

44

170

300

7 7.5 12

44

170

300

7 8.5 12

44

170

300

7 9.0 170

12 6.5 12

44

170

300

12 7.5 12

44

LC50 and 95% confidence interval i

3 h

1.79 

1.31-2.44

2.00 

1.74-2.29

2.60 

1.71-3.96

1.70 

1.27-2.28

>4.00 a

4.25 

3.43-5.26

7.60 

5.21-11.1

6.40 

5.55-7.39

20.7 

14.6-29.4

>15.0 a

29.5 

24.5-35.6

35.0 

27.6-44.4

54.0 

40.4-72.2

1.10 

0.847-1.43

> 0.600°

1.22 

0.996-1.49

1.25 

1.02-1.53

3.90 

2.66-5.71

2.50 

1.67-3.74

6h

1.20 

1.05-1.38

0.915 

0.773-1.08

1.38 

1.05-1.82

1.42 

1.15-1.76

5.60 

3.44-9.12

3.17 

2.64-3.81

7.60

5.21-11.1

6.30 

5.44-7.30

12.0 

10.4-13.8

>15.0 a

29.5 

24.5-35.6

28.5 

23.0-35.3

54.0 

40.4-72.2

0.740 

0.616-0.889

> 0.600°

1.22 

0.996-1.49

1.10 

0.929-1.30

1.85 

1.26-2.72

2.50 

1.67-3.74

12 h

0.545 

0.462-0.643

0.655 

0.518-0.829

0.560 

0.467-0.671

0.770 

0.587-1.01

1.53 

1.15-2.04

1.72 

1.48-2.00

3.12 

2.61-3.73

3.70 

2.83-4.83

7.80 

6.67-9.12

15.0 

11.6-19.4

13.1 

10.8-15.9

28.0 

22.6-34.7

26.1 

22.0-31.0

0.450 

0.343-0.590

0.640 

0.476-0.860

0.542 

0.460-0.639

0.780 

0.646-0.941

1.62 

1.30-2.02

2.50 

1.67-3.74

24 h

0.500 

0.319-0.783

0.225 

0.137-0.370

0.452 

0.335-0.611

0.390 

0.291-0.522

1.32 

1.00-1.73

1.72 

1.48-2.00

2.00 

1.64-2.43

2.38 

1.87-3.02

2.70 

2.03-3.58

10.4 

8.93-12.1

8.60 

7.37-10.0

10.2 

8.57-12.0

17.2 

14.8-20.0

0.420 

0.339-0.520

0.420 

0.339-0.520

0.490 

0.389-0.618

0.550 

0.465-0.651

1.40 

1.13-1.73

1.57 

1.20-2.05

f Ml/1) at

48 h

0.385 

0.294-0.504

0.225 

0.137-0.370

0.275 

0.181-0.418

0.332 

0.237-0.466

0.720 

0.509-1.02

1.30 

1.03-1.64

1.41 

1.14-1.74

1.75 

1.25-2.46

2.16 

1.27-3.66

7.80 

5.80-10.5

7.00 

5.66-8.66

8.60 

7.46-9.91

11.5 

8.82-15.0

0.420 

0.339-0.520

0.420 

0.339-0.520

0.490 

0.389-0.618

0.550 

0.465-0.651

1.40 

1.13-1.73

1.57 

1.20-2.05

96 h

0.330 

0.237-0.459

0.225 

0.137-0.370

0.275 

0.181-0.418

0.277 

0.209-0.367

0.600 

0.418-0.861

1.18 

0.906-1.54

1.41 

1.14-1.74

1.75 

1.25-2.46

1.95 

1.24-3.06

4.55 

3.43-6.03

6.10 

4.75-7.84

6.90 

5.47-8.70

11.0 

8.34-14.5

0.381 

0.290-0.500

0.420 

0.339-0.520

0.490 

0.389-0.618

0.550 

0.465-0.651

1.31 

0.997-1.72

1.57 

1.20-2.05
12

Appendix (Cont'd)

_ Total Temp. 

(0C\ P" hardness

( ' (mg/l CaC0 3 )

170

300

12 8.5 12

44

170

300

12 9.0 12

44

170

17 6.5 12

44

170

300

17 7.5 12

44

170

300

17 8.5 12

44

LC50 and 95% confidence interval (/Ill/I) at

3 h

4.62 

3.66-5.83

6.40 

5.49-7.46

8.40 

6.86-10.3

>5.00 a

22.9 

20.4-25.7

34.5 

27.2-43.8

42.0 

31.4-56.2

>30.0 a

36.5 

30.6-43.5

1.59 

0.985-2.57

>1.00 a

1.65 

1.24-2.20

1.71 

1.27-2.29

>2.50 a

>2.50 a

10.0 

6.35-15.7

12.7 

8.42-19.2

8.90 

7.16-11.0

>8.00 a

6 h

4.62 

3.66-5.83

3.85 

3.29-4.50

7.70 

6.38-9.29

>5.00 a

23.1 

19.9-26.8

38.5 

29.9-49.6

>30.0 a

...

0.820 

0.632-1.06

1.38 

0.800-2.38

0.850 

0.687-1.05

1.22 

0.993-1.50

2.05 

0.984-4.27

2.50 

1.75-3.57

4.25 

3.43-5.26

6.30 

5.46-7.27

8.81 

7.07-11.0

>8.00 a

12 h

2.90 

2.10-4.01

3.80 

3.09-4.68

5.40 

4.58-6.37

4.90 

3.51-6.84

13.6 

12.3-15.1

14.2 

11.9-16.9

33.0 

23.5-46.3

24.5 

21.3-28.2

22.2 

17.7-27.8

0.600 

0.495-0.727

0.560 

0.453-0.693

0.520 

0.434-0.623

0.840 

0.659-1.07

1.78 

1.51-2.09

1.91 

1.56-2.34

3.20 

2.68-3.82

6.20 

5.39-7.13

7.60 

5.81-9.95

11.3 

6.40-20.0

24 h

2.45 

2.04-2.94

2.83 

2.11-3.79

3.90 

3.00-5.08

4.90 

3.51-6.84

9.40 

7.81-11.3

11.6 

9.85-13.7

20.0 

16.4-24.3

20.0 

17.8-22.4

20.8 

17.0-25.5

0.455 

0.337-0.614

0.560 

0.453-0.693

0.519 

0.431-0.625

0.640 

0.460-0.890

1.23 

1.02-1.48

1.60 

1.27-2.02

2.80 

2.26-3.47

2.35 

1.86-2.97

4.60 

3.38-6.26

10.0 

5.85-17.1

48 h

2.04 

1.61-2.58

2.83 

2.11-3.79

3.28 

2.40-4.47

4.90 

3.51-6.84

8.60 

7.45-9.93

10.0 

8.19-12.2

17.1 

14.4-20.3

17.8 

15.7-20.2

18.0 

15.3-21.2

0.455 

0.337-0.614

0.560 

0.453-0.693

0.519 

0.431-0.625

0.620 

0.450-0.855

1.23 

1.02-1.48

1.60 

1.27-2.02

2.80 

2.26-3.47

2.30 

1.82-2.90

1.75 

1.25-2.46

6.80 

4.53-10.2

96 h

2.04 

1.61-2.58

2.83 

2.11-3.79

3.28 

2.40-4.47

4.35 

3.24-5.84

8.60 

7.45-9.93

10.0 

8.19-12.2

15.0 

11.6-19.4

17.2 

14.6-20.3

18.0 

15.3-21.2

0.455 

0.337-0.614

0.560 

0.453-0.693

0.519 

0.431-0.625

0.600 

0.439-0.820

1.23 

1.02-1.48

1.60 

1.27-2.02

2.80 

2.26-3.47

2.30 

1.82-2.90

1.75 

1.25-2.46

4.25 

3.43-5.26
13 

Appendix (Cont'd)

_ Total Temp. 

,or\ P" hardness

(mg/l CaCOa)

170

300

17 9.0 170

22 6.5 12

44

170

300

22 7.5 12

44

170

300

22 8.5 12

44

170

300

22 9.0 170

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

3 h

26.2 

22.1-31.1

3.15 

25.7-38.6

52.0 

42.7-63.3

0.900 

0.719-1.13

> 0.800°

1.40 

1.05-1.87

1.40 

0.935-2.10

>2.50°

>2.50°

>6.00°

5.41 

4.59-6.38

16.0 

10.0-25.6

>10.0

37.5 

29.1-48.4

26.0 

19.7-34.3

125.0 

74.2-2.11

6 h

26.2 

22.1-31.1

15.0 

13.4-16.8

52.0 

42.7-63.3

0.580 

0.474-0.710

0.800 

0.543-1.18

0.960 

0.785-1.17

1.40 

0.935-2.10

2.00 

1.64-2.43

1.87 

1.54-2.27

4.20 

3.39-5.20

3.85 

3.29-4.50

9.50 

7.52-12.0

12.2

8.24-18.1

17.0 

15.3-18.8

15.6 

13.8-17.7

46.6 

36.7-59.2

12 h

18.9 

16.4-21.8

11.7 

10.1-13.5

33.0 

26.7-40.8

0.460 

0.308-0.687

0.560 

0.453-0.693

0.860 

0.690-1.07

0.860 

0.639-1.16

1.90 

1.62-2.24

1.87 

1.54-2.27

4.20 

3.39-5.20

3.85 

3.29-4.50

4.60 

3.08-6.87

9.40 

6.82-13.0

16.2 

14.1-18.6

12.2 

10.8-13.8

40.0 

33.0-48.5

24 h

14.4 

13.0-15.9

8.60 

7.45-9.93

28.0 

22.6-34.7

0.450 

0.364-0.557

0.560 

0.453-0.693

0.860 

0.690-1.07

0.860 

0.639-1.16

1.25 

1.09-1.43

1.87 

1.54-2.27

3.65 

3.06-4.35

3.35 

2.62-4.28

4.20 

3.14-5.62

8.20 

6.71-10.0

15.2 

13.5-17.2

10.0 

8.27-12.1

37.5 

31.2-45.0

48 h

14.4 

13.0-15.9

8.60 

7.45-9.93

28.0 

22.6-34.7

0.450 

0.364-0.557

0.560 

0.453-0.693

0.760 

0.628-0.919

0.860 

0.639-1.16

1.25 

1.09-1.43

1.84 

1.52-2.22

3.65 

3.06-4.35

2.51 

1.95-3.23

4.20 

3.14-5.62

8.20 

6.71-10.0

15.2 

13.5-17.2

9.00 

7.19-11.3

37.5 

31.2-45.0

96 h

14.4 

13.0-15.9

8.60 

7.45-9.93

28..0 

22.6-34.7

0.450 

0.364-0.557

0.560 

0.453-0.693

0.760 

0.628-0.919

0.860 

0.639-1.16

1.25 

1.09-1.43

1.84 

1.52-2.22

3.46 

2.97-4.04

2.51 

1.95-3.23

4.20 

3.14-5.62

8.20 

6.71-10.0

15.2 

13.5-17.2

8.70 

7.47-10.1

37.5 

31.2-45.0

'No mortality at highest concentration tested.

INVESTIGATIONS IN FISH CONTROL

64. Effects of 3-Trifluoromethyl-4-nitrophenol

(TFM) on Developmental Stages of the Sea Lamprey

By George W. Piavis and John H. Howell

United States Department of the Interior

Fish and Wildlife Service 

Washington, B.C. 1975
CONTENTS

Page

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

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

Materials and methods.................................................. 4

Effects of exposure to TFM on embryonic development .................. 4

Implications of the experiment .......................................... 8

Literature cited ......................................................... 8
EFFECTS OF 3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM) 

ON DEVELOPMENTAL STAGES OF THE SEA LAMPREY1

by

George W. Piavis2 and John H. Howell 

Hammond Bay Biological Station, Millersburg, Michigan

ABSTRACT

Developing sea lampreys (Petromyzon marinus) in stages 1 (zygote) through 17 

(burrowing prolarva) were exposed for 24 h to a 10-mg/l (active ingredient) solution 

of 3-trifluoromethyl-4-nitrophenol (TFM) at 18 C. Embryonic development, 

incidence of abnormalities, and mortality in the experimentals were compared with 

those in unexposed controls. Although exposed embryos in the first eight 

developmental stages exhibited no immediate effects, the number of viable stage-18 

larvae produced was drastically reduced, incidence of abnormalities increased 

markedly, hatching was sometimes delayed, and hemoglobin production was 

retarded or lacking. The laboratory findings suggest that the treatment of streams 

with TFM at the customary rates probably does not effect a complete kill of sea 

lampreys in all developmental stages.

INTRODUCTION

The selective lampricide 3-trifluoromethyl4-nitrophenol (TFM), used in tributaries of the 

Great Lakes to control the sea lamprey 

(Petromyzon marinus), is toxic to lampreys from 

stage-18 larvae (Piavis 1962) to spawning 

adults (Applegate et al. 1961). Its toxicity to 

earlier developmental stages of the sea lamprey, 

however, has not been determined. Piavis 

(1962), who subjected several developmental 

stages to six selective lamprey larvicides 

TFM, four other nitrophenols, and a 

thiocarbamate showed that some lampreys 

survived exposure to a 10-mg/l concentration at 

stages 10 (neural plate and groove) or 13-14

This investigation was supported in part by the 

National Institute of Health Research Grants 

GM-09836-04 and FR-05317-06 and by the Great 

Lakes Fishery Commission.

2Alternate address: Department of Anatomy, 

School of Dentistry, University of Maryland, 

Baltimore, Maryland 21201.

(prehatching and hatching), but not at certain 

other stages: 8 (blastula), 9 (gastrula), and 

during transition from stage 17 (burrowing 

prolarva) to stage 18 (larva). He concluded that 

chemical treatment of streams with larvicides 

would be most effective if carried out at least 40 

days after all spawning ceased. Forty days is 

the longest approximate time required for 

lamprey development to progress from fertilization to stage 18 (Piavis 1961).

The spawning season of the sea lamprey in 

the upper Great Lakes usually lasts about 2 mo, 

June and July, and development of the last 

embryos extends through August. Stream 

treatments during these months are facilitated, 

however, by favorable physical and biological 

factors such as low stream flow, suitable water 

temperature, and acceptable biological activity 

of TFM. Consequently it was highly desirable to 

determine whether TFM is toxic to sea lamprey 

embryos at all stages of development.
MATERIALS AND METHODS

Gametes were obtained from spawning pairs 

of sea lampreys collected in the Ocqueoc River, 

Presque Isle County, Michigan. Two batches of 

eggs were fertilized by the procedures described 

by Piavis (1961); one male and one female were 

used for each batch. Fertilized eggs were 

apportioned among 18-cm glass bowls that 

were immersed in water in 10-liter battery jars. 

The bath water in each jar consisted of 6 to 7 

liters of filtered Lake Huron water, tempered to 

18 C in constant-temperature troughs. Each 

bowl was aerated by a stone air breaker.

The developmental stages and their distinguishing features, and (in parentheses) the 

time between the first and last appearance of the 

stage in samples reared at 18 C (from Piavis 

1961) are as follows: stage 0, ovulated but 

unfertilized ovum; 1, zygote (0-2 h); 2,2 cells (2-8 

h); 3, 4 cells (8-11 h); 4,8 cells (10-15 h); 5,16 cells 

(13-15 h); 6, 32 cells (16-19 h); 7, 64 cells (19-24 

h); 8, full blastula (24-64 h); 9, gastrula (64-104 

h); 10, neural plate and groove (4-5 days); 11, 

neural rod (5-6 days); 12, head distinguishable 

(6-8 days); 13, prehatching (8-12 days); 14, 

hatching (10-13 days); 15, pigmentation (13-16

days); 16, gill clefts (15-17 days); 17, burrowing 

larva (17-33 days); and 18, larva (33-40 days).

As animals in each stage of development 

became successively available, they were 

removed from one of the two batches, counted, 

and exposed for 24 h to a 10-mg/l (active 

ingredient) solution of TFM at 18 C. Exposures 

were made in a glass bowl and battery jar 

combination like that previously described. The 

remaining embryos served as controls. After 

exposure, the embryos were washed and 

transferred to fresh water. Further development, abnormalities, and mortalities of the 

exposed embryos were compared with those of 

the controls on the basis of periodic sample 

counts. Abnormal embryos which were so 

grossly deformed that no precise stage could be 

assigned to them were counted as dead in the 

final samples. Terminal samples were taken at 

stage 17 for all lampreys that reached this stage; 

the living lampreys (normal and abnormal) 

were then held long enough to allow them to 

advance to stage 18. All terminal-sample 

percentages are cumulative from the egg 

through the indicated stage.

EFFECTS OF EXPOSURE TO TFM 

ON EMBRYONIC DEVELOPMENT

Embryonic development and mortality were 

roughly similar in the two batches of eggs used 

as controls. Ofihe 6,686 fertilized eggs obtained 

from one pair of lampreys, 2,747 were exposed to 

TFM at stages 1-7 and 17; survival to normal 

stage 18 among the remainder (controls) was 

68% (Table 1). Of the 4,616 fertilized eggs 

obtained from the second pair, 2,107 were 

exposed to TFM at stages 8-16, survival to 

normal stage 18 among the remainder was 75%.

The effects of exposure of embryos and 

prolarvae (stages 15-17) to 10-mg/l TFM (a.i.) 

for 24 h varied considerably with the stage of 

development at the time of exposure. Embryos 

in stages 1-7 advanced to successive stages 

while they were being exposed to the 24-h 

treatment. More advanced stages (8-17) did not, 

primarily because the duration of the stage 

normally would be expected to exceed the 24-h 

exposure period.

The principal results, listed according to stage 

at the time of exposure, are summarized below 

and in Table 1.

Stage 1 Normal development through stage 

12; abnormalities developed during stage 13; 

mortality increased progressively to 100% 

during stage 17.

Stage 2 Normal development through stage 

9; abnormalities and mortalities were significant during stage 10, and increased progressively to 100% during stage 17.

Stage 3 Normal development through stage 

12; abnormalities and mortalities became 

significant during stage 13; mortality reached 

81% during stage 17. Of the 369 embryos in the 

lot, 70 (19%) yielded normal stage-18 larvae.

Stage 4 Normal development through stage 

12; abnormalities and mortalities significantly 

increased during stage 13; mortalities increased 

progressively to 100% during stage 17.
Table 1. Effect of TFM on developing sea lampreys: percentage of normal stage-18 larvae 

produced after lampreys were exposed at different developmental stages to 10-ppm TFM 

for 24 h at 18.4 C

Stage at 

exposure

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Total

Control 1 °

Control 2°

Number 

exposed

354

275

369

199

286

603

611

275

246

252

212

314

200

203

245

160

50

4,854

3,939

2,509

Most advanced 

stage attained

17

17

18

17

18

17

18

17

16

17

16

16

17

15

15

16

17

18

18

Percentage 

of surviving stage- 18 

larvae

0

0

19.0

0

1.4

0

5.7

0

0

0

0

0

0

0

0

0

0

2.2

68.1

74.9

1 Lampreys exposed at developmental stages 1 -7 and 17 were from the same parents as those from Control 1; the animals exposed at 

stages 8-16 were from the same parents as those from Control 2.

Stage 5 Normal development through stage 

9; abnormalities appeared during stage 11; 

mortalities reached 48% during stage 13 and 99% 

during stage 17. Of the 286 embryos in the lot, 4 

(1%) advanced to normal stage-18 larvae.

Stage 6 Normal development through stage 

12; mortality reached 68% during stage 13; 

abnormalities increased to 75% of the survivors 

at stage 15; mortalities increased progressively 

to 100% during stage 17.

Stage? Normal development through stage 

9; mortality was 27% at stage 10 and 94% at 

stage 17. Of the 611 embryos in this lot, 35 (6%) 

survived to become normal stage-18 larvae.

Stage 8 Normal development through stage 

13; mortality during exposure, 18%; abnormalities in 45% of the specimens through stage 

16; mortality reached 100% during stage 17.

Stage 9 Mortality during exposure, 18%; 

abnormalities appeared at stage 10; mortality 

increased progressively to 100% during stage 17.

Stage 10 No mortality during exposure; 

abnormalities increased to 49% at stage 15; 

mortality reached 21% at stage 15 and increased 

to 100% at stage 17.

Stage 11 Mortality during exposure, 19%; 

abnormalities increased to 87% and mortality 

reached 60% at stage 16; mortality reached 100% 

during stage 16.

Stage 12 Mortality was 21% during exposure, and reached 100% during stage 16.

Stage 13 Mortality 19% during exposure; 

100% abnormalities after exposure; mortality 

reached 100% during stage 17.
Stage 14 Mortality 89% in stage 14, after 

exposure; mortality reached 100% during stage 

15.

Stages 15, 16, and 17 Total mortality 

during exposure.

Lamprey embryos exposed to TFM during the 

early stages of development (1-8) exhibited no 

immediate effects; usually the effects were 

delayed until stage 13. The embryos either 

attained stage 8 during the exposure period or, if 

exposed at stage 8, remained at that stage; the 

time sequence of development of the embryos 

exposed at these stages was thus identical with 

that for the controls and within the normal 

range reported by Piavis (1961). Embryos 

exposed to TFM during stages 8-14 remained in 

the stage of exposure during exposure; all 

prolarvae in stages 15-17 died during exposure 

(Table 1). Thus, except for a few normal larvae 

that developed after exposure within 24 h after 

fertilization, at stages 3, 5, and 7, all embryos 

and prolarvae exposed to TFM became grossly 

abnormal or died.

Exposure of lampreys, at any stage of development, to 10-mg/l TFM (a.i.) for 24 h greatly 

increased the incidence of abnormalities. There 

seemed to be no marked distinction between 

stage 1 through 8 and stages 9 through 17; 

abnormalities in lots exposed at each stage far 

exceeded the rate in the controls. The number of 

abnormal larvae that reached stage 18 after 

exposure at different stages were as 

follows: exposure at stage 3, 95; stage 5, 41; 

stage 7, 15; stage 8, 6; and stage 10, 10. 

Abnormal stage-18 larvae in the controls 

numbered 271 in Control No. 1 and 61 in Control 

No. 2.

Although many of the embryos exposed 

during cellular stages (1-7) continued development and eventually hatched, they failed to do 

so at the normal stage (14). Development within

the egg, however, did not cease. Elongation of 

the embryo continued, resulting in curling and 

coiling within the chorionic space, until the 

embryo completely filled it. Pigment spots, 

heart beat, velum movement, and open gill slits 

(all characteristics of stage-16 embryos) were 

often present in extreme cases of "delayed stage 

13's." Although most of these embryos eventually hatched, they were grossly deformed, usually 

into the shape of the letters C, J, P, or O (Fig. 1). 

Such deformed animals did not straighten after 

hatching, never swam normally, and were 

unable to burrow.

Normal sea lamprey embryos initiate synthesis of hemoglobin during stage 15, as 

indicated by the reddish cast of the blood or the 

reddish color of the red blood cells under high 

magnification. In the present study, however, 

hemoglobin was not detected at this stage, in 

any of the exposed embryos confirming 

similar findings by Piavis (1962) after he 

exposed sea lamprey embryos to several 

halogenated nitrophenols. Apparently tht' lack 

of hemoglobin inhibited further development in 

most embryos. The nearly normal velum 

movements and heart beats of the exposed 

embryos that attained stage 16 decreased 

greatly with passage of time within that stage. 

Movement diminished to the extent that prodding with a teasing needle produced only 

localized body twitches. The gallbladder 

became, light green and was greatly distended. 

Although a few embryos eventually developed 

eyespots (a criterion for stage 17), they were 

unable to burrow. Even the few embryos that 

reached stage 18 in normal condition (after 

exposure at stages 3, 5, and 7) appeared to have 

lower than normal hemoglobin levels, had 

abnormally large gallbladders, and moved 

sluggishly. These symptoms disappeared, 

however, as development continued in stage 18.
Figure 1. Some 

- ' 

of the more prominent changes in body contour effected by exposure of developmental stages 

W "°" ShaPed Pr°lttrVa' <b> "J" ****** *rO1" ' <°> "°" «
IMPLICATIONS OF THE EXPERIMENT

Although production of viable stage-17 probably do not kill all sea lamprey embryos,

prolarvae was very markedly reduced, from 71% Some embryos less than 24 h old at the time of

in the controls to only 4% after exposure of treatment would be expected to survive (at water

stages 1-7 to 10-mg/l TFM and nil after temperatures near 18 C), and the percentage of

exposure of stages 8-17, this experiment in- survivors would be expected to increase as

dicates that treatments of streams with TFM concentration and time of exposure decreased

during the sea lamprey spawning season from 10 mg/1 and 24 h.

LITERATURE CITED

Applegate, V. C., J. H. Howell, J. W. Moffett, B. G. H. Piavis, G. W. 1962. Exposure of several developmental 

Johnson, and M. A. Smith. 1961. Use of 3-trifluormeth- stages of the sea lamprey, Petromyzon marinas, to 

yl-4-nitrophenol as a selective sea lamprey larvicide. selective larvicides. Copeia 1962(3):652-653. 

Great Lakes Fish. Comm., Tech. Rep. 1. 35 pp.

Piavis, G. W. 1961. Embryological stages in the sea lamprey 

and effects of temperature on development. U.S. Fish 

Wildl. Serv. Fish. Bull. 61:111-143.
INVESTIGATIONS IN FISH CONTROL

65. Accumulation and Loss of Residues of 

3-Trifluoromethyl-4-nitrophenol (TFM) 

in Fish Muscle Tissue: Laboratory Studies

By Joe B. Sills and John L. Alien

United States Department of the Interior

Fish and Wildlife Service 

Washington, D.C. 1975
CONTENTS

Page

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

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

Materials and methods.................................................. 4

Accumulation and loss of TFM residues in eight species of fish ........ 4

Rainbow trout ...................................................... 4

Channel catfish..................................................... 4

Other species of fish ................................................ 8

Discussion.............................................................. 9

Conclusions............................................................. 10

Literature cited ......................................................... 10
ACCUMULATION AND LOSS OF RESIDUES OF

3-TRIFLUOROMETHYL-4-NITROPHENOL(TFM)

IN FISH MUSCLE TISSUE: LABORATORY STUDIES

by

Joe B. Sills

Southeastern Fish Control Laboratory 

Warm Springs, Georgia

and

John L. Alien 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT

Residues of 3-trifluoromethyl-4-nitrophenol (TFM) in muscle tissue of eight 

species offish, after they were exposed under controlled conditions, were determined 

by gas chromatography. The mean concentration of TFM residue in samples from 

various species immediately after a 12-h exposure to 1 to 4 mg/1 of TFM ranged from 

0.02 to 5.09 fjig/g depending on pH, temperature, hardness of the test solutions, and 

TFM concentration. Residues decreased rapidly after the fish were withdrawn from 

the test media, and were near the limit of detection (0.01 /-tg/g) within 24 h.

INTRODUCTION

Control of the parasitic sea lamprey The concentration of TFM residues in fish

(Petromyzon marinus) in the Great Lakes is exposed to TFM and the persistence of the

dependent on the application of selective tox- residues is part of the safety information

icants to streams inhabited by the larvae. The required to retain its registration. In the present

toxicant that has been most widely used is study we measured the concentration and

3-trifluoromethyl-4-nitrophenol (TFM). To re- persistence of TFM residue in fish muscle after

tain the registered use of TFM, the U.S. exposure of eight species of fish to measured

Environmental Protection Agency requires that quantities of TFM. 

its safety for use in natural waters be evaluated.
MATERIALS AND METHODS

Eight species of fish representative of Great 

Lakes populations were exposed to selected 

concentrations of field grade TFM (39.4% 

sodium salt with 35.7% free phenol) for 12 h in 

100-liter polyethylene tanks with aeration. The 

12-h exposure period (usually to a TFM concentration of 1 mg/1) was used to simulate the time 

that fish are commonly exposed during stream 

treatments. After each test the fish were placed 

in a flow of fresh water of the same temperature 

for recovery. Concentrations were calculated on 

the free phenol basis. Water hardness, pH, and 

temperature were varied in some tests to 

evaluate their effects on TFM uptake and 

residue elimination. The hardness and pH of the 

water used during the exposure periods were 

controlled as described by Marking and Dawson 

(1973). Fish weighing 100 g or more were used in 

all tests to ensure enough muscle tissue for 

analysis. Samples consisting of five fish each 

were selected for analysis usually at intervals of 

4 h for 24 h after transfer to fresh water. Fish 

were filleted and frozen immediately after 

selection.

Frozen fish samples were prepared for 

analysis by the method of Benville and Tindle 

(1970) and extracted by the column technique of 

Hesselberg and Johnson (1972). The fish extracts were analyzed by the gas chromatographic method of Alien and Sills (1974). The 

method is capable of detecting 0.01 ng of TFM; 

samples containing less than 0.01 ji g/g of TFM 

residue are reported as 0.00.

Rainbow trout (Salmo gairdneri), brown trout 

(S. trutta), lake trout (Salvelinus namaycush), 

carp (Cyprinus carpio), and white bass (Morone 

chrysops) were treatec^ and sampled at the Fish 

Control Laboratory, La Crosse, Wis. Frozen 

fillets were sent to the Southeastern Fish 

Control Laboratory, Warm Springs, Ga. for 

analysis. The channel catfish (Ictalurus punctatus), bluegill (Lepomis macrochirus), and 

largemouth bass (Micropterus salmoides) were 

treated, sampled, and analyzed at the 

Southeastern Fish Control Laboratory. We 

tested rainbow trout, representing the coldwater 

species, and channel catfish, representing the 

warmwater species, more extensively than the 

other species to determine effects of 

temperature, hardness, concentration of TFM,

and pH on residue accumulation and elimination.

Accumulation and Loss of TFM Residues 

in Eight Species of Fish

Rainbow Trout

Rainbow trout ranging in weight from 100 to 

600 g were exposed to 1-mg/l solutions of TFM in 

waters adjusted to four different hardnesses and 

three different temperatures (Table 1). 

Temperature changes per se had relatively little 

effect on the accumulation and elimination of 

TFM residue. The mean residue levels immediately after the 12-h exposure ranged from 

0.30 to 1.01 /u.g/g, and showed no consistent 

change with different temperatures. Residues 

were near the lowest detectable limit of 0.01 

fj,g/g 24 h after withdrawal in all three tests.

Increases or decreases in hardness, which 

were accompanied by corresponding changes in 

pH, produced noticeable differences in the 

accumulation and elimination of TFM residue 

when temperature was held constant. The mean 

concentration of TFM residue at withdrawal 

was lowest in fish from the hardest water (0.11 

/ug/g) and highest in the softest water (2.77 

/Ag/g). The rates of accumulation and elimination were both greatest among fish in the softest 

water tested; and the slightly higher residue 

level remaining after a 24-h withdrawal 

probably resulted from the much higher initial 

accumulation. The residues after 24 h in the 

other tests were 0.00 or 0.01 j/g/g.

Channel Catfish

Channel catfish weighing from 400 to 1,200 g 

were exposed to 1-mg/l solutions of TFM 

adjusted to three different temperatures and 

four different water hardnesses (Table 2), and to 

solutions containing 4 mg/1 of TFM at three 

temperatures in water having a hardness of 160 

to 180 mg/1 (Table 3). As in rainbow trout, 

accumulation and elimination of TFM residue 

were not closely related to temperature. Fish 

exposed to 4-mg/l concentrations of TFM 

accumulated more residue than those exposed to 

1-mg/l concentrations. The elimination of 

residue, however, followed the pattern described 

for rainbow trout, and the TFM concentration
Table 1. Residues of TFM in rainbow trout exposed to 1-mg/l concentrations of TFM for 12 h

Test solutions Withdrawal

Hardness Temp. interval 

(as mg/l CaCO3 ) P (°C) (hours)

10-13 6.4-6.8 12 Control

0

4

12

24

40-48 7.2-7.6 7 Control

0

12

24

40-48 7.2-7.6 12 Control

0

4

8

12

40-48 7.2-7.6 17 Control

0

8

24

160-180 7.6-8.0 12 Control

0

4

8

12

24

280-320 8.0-8.4 12 Control

0

4

8

12

24

TFM residue

Mean

0.00

2.77

1.42

0.11

0.04

0.00

1.01

0.19

0.01

0.00

0.30

0.02

0.02

0.01

0.00

0.74

0.03

0.01

0.00

0.15

0.03

0.05

0.01

0.01

0.00

0.11

0.04

0.00

0.00

0.00

(M9/9)°

Range

0.00-0.01

2.40-3.10

1.30-1.55

0.08-0.12

0.02-0.05

0.00-0.00

0.74-1.31

0.02-0.53

0.01-0.02

0.00-0.00

0.01-0.79

0.01-0.03

0.00-0.06

0.01-0.01

0.00-0.00

0.60-1.07

0.01-0.06

0.01-0.01

0.00-0.00

0.04-0.27

0.02-0.05

0.02-0.09

0.01-0.02

0.01-0.02

0.00-0.00

0.05-0.23

0.01-0.10

0.00-0.01

0.00-0.00

0.00-0.00

Each mean and range represents five analyses.
Table 2. Residues of TFM in channel catfish exposed to 

1-mg/l concentrations of TFM for 12 h

Test solutions Withdrawal

Hardness Temp. interval 

(as mg/l CaCO3 ) P (°C) (hours)

20-22 7.1-7.6 12 Control

0

4

8

12

24

20-22 7.1-7.6 18.5 Control

0

4

8

12

24

20-22 7.1-7.6 27 Control

0

4

8

12

24

40-48 7.2-7.6 18.5 Control

0

4

8

12

24

160-180 7.6-8.0 18.5 Control

0

4

8

12

24

280-320 8.0-8.4 18.5 Control

0

4

8

12

24

TFM residue (

Mean

0.00

1.75

0.67

0.32

0.07

0.02

0.00

1.67

0.18

0.06

0.02

0.02

0.00

1.41

0.03

0.01

0.01

0.01

0.00

0.77

0.10

0.02

0.01

0.00

0.00

0.33

0.02

0.00

0.00

0.00

0.00

0.13

0.01

0.00

0.00

0.00

M9/9>°

Range

0.00-0.00

1.10-2.20

0.45-1.04

0.08-0.70

0.03-0.10

0.01-0.04

0.00-0.00

1.10-2.40

0.10-0.26

0.02-0.10

0.00-0.05

0.00-0.06

0.00-0.00

1.00-2.00

0.03-0.04

0.01-0.01

0.01-0.02

0.00-0.01

0.00-0.00

0.64-0.93

0.07-0.12

0.01-0.03

0.01-0.01

0.00-0.00

0.00-0.00

0.16-0.60

0.01-0.03

0.00-0.01

0.00-0.00

0.00-0.00

0.00-0.00

0.01-0.30

0.00-0.02

0.00-0.01

0.00-0.00

0.00-0.00

' Each mean and range represents five analyses.
Table 3. Residues of TFM in channel catfish exposed to 

4-mg/l concentrations of TFM for 12 h

Test solutions

Hardness 

(mg/l as CaCO3 ) P

160-180 7.6-8.0

160-180 7.6-8.0

160-180 7.6-8.0

Withdrawal

Temp. interval

(°C) (hours)

12 Control

0

4

8

12

24

17 Control

0

4

8

24

27 . Control

0

4

8

12

24

TFM residue (

Mean

0.00

5.09

2.28

0.33

0.66

0.04

0.00

2.09

0.64

0.39

0.03

0.00

2.59

0.10

0.03

0.01

0.01

A/9/9) a

Range

0.00-0.00

2.07-8.60

0.80-4.60

0.19-0.62

0.20-1.28

0.01-0.09

0.00-0.00

1.90-2.53

0.36-0.92

0.08-1.12

0.02-0.04

0.00-0.00

2.07-3.17

0.07-0.12

0.02-0.05

0.01-0.02

0.00-0.04

'Each mean and range represents five analyses.

was only slightly above the background concentration after 24 h. The elimination of TFM was 

slightly accelerated at the highest temperature. 

In fish exposed to a 1-mg/l concentration of 

TFM, the mean residue at withdrawal ranged 

from 0.13 to 1.75 //g/g immediately after 

withdrawal and from 0.00 to 0.02 //g/g after 24 

h of withdrawal. In fish exposed to the higher 

concentration, these values ranged from 2.09 to 

5.09 ng/g at withdrawal and from 0.01 to 0.04 

H g/g after 24 h of withdrawal.

The accumulation of TFM again was closely 

related to the hardness of the test medium. As in 

rainbow trout, channel catfish accumulated the 

highest level of TFM residue in the softest test 

solution. However, the elimination was essentially complete after 24 h in all tests.

In tests of the effect of pH on the accumulation 

of TFM in channel catfish the temperature, 

hardness, and concentration of TFM were held

Table 4. Residues of TFM in channel catfish 

muscle tissue immediately after exposure 

to a 1-mg/l concentration of TFM for 12 h 

at 18.5 C, a water hardness of 45 mg/l as 

CaCO3 , and various pH's

pH

6

7

8

9

Mean 0

3.21

1.53

0.33

0.03

j/g/g of TFM in muscle

95% confidence interval

0.00-6.51

1.22-1.84

0.29-0.37

0.03-0.05

a Each mean and range represents five analyses.
constant in solutions that were buffered to pH's 

6, 7, 8, and 9 (Table 4). The analysis of fish 

immediately after withdrawal clearly showed 

that the uptake of TFM decreased as pH 

increased. The mean concentration of TFM was 

3.21 !/g/g at pH 6 and 0.03 ^g/g at pH 9. The 

decrease was 10-fold from pH 6 to pH 8 and 

100-fold from pH 6 to pH 9. The pH variation 

between the unbuffered test solutions was the 

most probable cause of TFM residue variation 

rather than increases in hardness per se.

Other Species of Fish

Persistence of TFM in muscle tissue of other 

fish (Table 5) was similar to that in rainbow 

trout and channel catfish. TFM residue was 

essentially eliminated from fish muscle after 24 

h in fresh water even in brown trout and carp, 

which retained low concentrations slightly 

longer than the other species.

Table 5. Residues of TFM in muscle tissue of six species of fish exposed to 

a 1-mg/l concentration of TFM for 12 h

Test solutions Withdrawal

Hardness Temp. interval 

(mg/l as CaCO3 ) P (°C) (hours)

Brown trout

40-48 7.2-7.6 12 , Control

Q

4

8

12

24

40-48 7.2-7.6 7 Control

0

4

8

24

Lake trout

40-48 7.2-7.6 12 Control

0

4

8

12

24

Carp

40-48 7.2-7.6 12 Control

0

4

8

12

24

White bass

40-48 7.2-7.6 12 Control

0

4

8

12

24

TFM residue

Mean

0.00

0.77

0.35

0.25

0.13

0.10

0.00

0.35

0.04

0.04

0.03

0.00

0.11

0.04

0.02

0.02

0.00

0.00

1.69

0.78

0.34

0.24

0.06

0.00

0.02

0.02

0.01

0.00

0.00

(i/g/g) a

Range

0.00-0.00

0.54-0.92

0.15-0.63

0.07-0.35

0.04-0.22

0.06-0.14

0.00-0.00

0.24-0.63

0.02-0.06

0.01-0.12

0.02-0.06

0.00-0.00

0.07-0.14

0.02-0.04

0.01-0.03

0.01-0.02

0.00-0.01

0.00-0.00

1.40-1.90

0.45-1.00

0.27-0.53

0.13-0.33

0.02-0.07

0.00-0.00

0.02-0.02

0.01-0.04

0.00-0.01

0.00-0.00

0.00-0.00
Table 5 Continued

Test solutions

Hardness Temp, 

(mg/l as CaCO3 ) P (°C)

Bluegill

20-22 6.5-6.9 18.5

Largemouth bass

20-22 6.5-6.9 18.5

Withdrawal 

interval

(hours)

Control

0

4

8

12

24

Control

0

4

8

12

24

TFM residue (

Mean

0.00

0.21

0.07

0.04

0.04

0.01

0.00

0.32

0.01

0.00

0.00

0.00

:«/g>-

Range

0.00-0.00

0.18-0.26

0.03-0.10

0.03-0.06

0.01-0.13

0.01-0.02

0.00-0.00

0.16-0.57

0.01-0.01

0.00-0.01

0.00-0.01

0.00-0.00

Each mean and range respresents five analyses.

DISCUSSION

Residues of TFM do accumulate in the muscle 

of fish, and the amount varies with species and 

exposure conditions. Though there was wide 

variation in residue concentrations immediately after exposure, the concentration decreased 

rapidly after the fish had been placed in fresh 

water. After 24 h of withdrawal in fresh water 

the TFM residues were almost completely 

eliminated (less than 0.01 to 0.04 0g/g). The 

factors which have the greatest influence on 

uptake of TFM are concentration and pH of the

medium. High pH has the effect of lowering the 

concentration of available TFM which lowers 

uptake. An increase in pH from 6 to 9 caused a 

100-fold reduction in TFM uptake in channel 

catfish. Water hardness seems to influence 

uptake, but may in fact be a result of pH changes 

because elevated pH accompanies increased 

hardness. Temperature affects fish activity and 

metabolism, and thus probably exerts some 

influence on residue uptake and elimination.
10

CONCLUSIONS

1. The concentration of TFM residues in the 

muscle of fish exposed to 1.0 to 4.0 mg/1 of 

TFM for 12 h ranged from 0.02 to 5.09 j*g/g; 

however the chemical disappeared almost 

completely within 24 h after withdrawal.

2. The accumulation of TFM residue in fish was

more dependent upon pH than on water 

hardness or temperature.

3. The rate of elimination of TFM residue 

increased slightly as the temperature was 

increased.

LITERATURE CITED

Alien, J. L., and J. B. Sills. 1974. GLC determination of 

3-trifluoromethyl-4-nitrophenol (TFM) residue in fish. J. 

Assoc. Off. Anal. Chem. 57(2):387-388.

Benville, P. E., Jr., and R. C. Tindle. 1970. Dry ice 

homogenization procedure for fish samples in pesticide 

residue analysis. J. Agric. Food Chem. 18(5):948-949.

Hesselberg, R. J., and J. L. Johnson. 1972. Column

extraction of pesticides from fish, fish food, and mud. Bull.

Environ. Contam. Toxicol. 7(2/3):115-120. 

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

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

Fish Control No. 48. 8 pp.
INVESTIGATIONS IN FISH CONTROL

66. Residues of 3-Trifluoromethyl-4-nitrophenol 

(TFM) in a Stream Ecosystem after Treatment 

for Control of Sea Lampreys

By Philip A. Gilderhus, Joe B. Sills, and John L. Alien

United States Department of the Interior

Fish and Wildlife Service 

Washington, B.C. 1975
CONTENTS

Page

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

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

Materials and methods.................................................. 4

Results and discussion .................................................. 5

References .............................................................. 7
RESIDUES OF 3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM)

IN A STREAM ECOSYSTEM AFTER TREATMENT

FOR CONTROL OF SEA LAMPREYS

by

Philip A. Gilderhus, Joe B. Sills, and John L. Alien

Fish Control Laboratories, La Crosse, Wisconsin, and

Warm Springs, Georgia

ABSTRACT

Samples of water, bottom soil, plants, invertebrates, and fish for residue analysis 

were collected from two stations on the East Au Ores River in Michigan before, 

during, and after treatment of the stream with 3-trifluoromethyl-4-nitrophenol 

(TFM) for control of sea lampreys (Petromyzon marinus). The residues were highest 

in samples collected as the last portion of full-strength TFM flowed past each station, 

and were much higher in water and organisms than in the bottom soil. Fish retained 

higher residues than other organisms 24 h after treatment (up to 6 //g/g); however, 

the residues decreased to less than 0.08 //g/g at 96 h after treatment. Residues in soil 

were among the lowest found in all samples collected during the study.

INTRODUCTION

The first experimental stream treatments 

with the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) for the control of sea lampreys 

(Petromyzon marinus) in the Great Lakes were 

conducted in the spring and summer of 1958 

(Applegate et al. 1961), and operational 

treatments began in the fall (Great Lakes 

Fishery Commission 1958). Since then, most of 

the tributaries to the Great Lakes which harbor 

sea lamprey larvae have been treated with TFM 

and some streams have been treated several 

times.

The concentration and exposure time required 

for each stream are determined in on-site 

bioassays conducted just before treatment 

(Howell and Marquette 1962). Treatment crews 

apply TFM to a stream at various points along 

the section to be treated to ensure the 

maintenance of lamprey-killing concentrations 

and exposure times.

Concern about the possible accumulation of 

TFM in the environment led to early studies to 

assess the residues. Billy et al. (1965) determined 

concentrations in water from streams during 

and after treatment, but were unable to recover

TFM from fish exposed to field-use concentrations in stream treatments or in raceway 

tests. They were, however, able to detect 

residues in fish exposed to exceptionally high 

concentrations. Kempe (1973) documented the 

decay rate of TFM in static water-soil systems 

under laboratory conditions.

Recent environmental protection legislation 

has prescribed stricter requirements for 

registration of pest-control chemicals and 

periodic review of existing registrations. During 

review of the TFM registration in 1970, the 

determination was made that the existing data 

on TFM residues were inadequate to satisfy the 

requirements of the Federal Insecticide, 

Fungicide, and Rodenticide Act.

The present study was undertaken in 

response to the need for further information on 

the persistence of TFM residues in representative components of a stream environment. 

Fish, invertebrates, water, plants, and bottom 

soil were sampled at selected intervals during 

and after an operational stream treatment for 

eradication of sea lamprey larvae, and analyzed 

for residues of TFM.
MATERIALS AND METHODS

The East Au Gres River in losco and Arenac 

counties, Michigan, was chosen for sampling 

because it is a fertile and productive stream 

inhabited by a diversity of organisms. The 

stream is about 40 km (25 mi) long, and has flow 

volumes of 0.57 to 0.71 mVsec (20-25 cfs) in its 

middle reaches and about 1.43 mVsec (50 cfs) 

near the mouth. The stream has a moderate 

gradient and extensive riffle areas.

The stream treatment of 25-26 July 1972 was 

designed to maintain 11 mg/1 of TFM for about 

12 h at any given point in the stream. Treatment 

was started in the headwaters and the concentration was boosted at selected locations 

downstream to compensate for dilution that 

resulted from increases in volume of flow 

between application points. The lampricide was 

applied at each site for several hours by means 

of battery-powered fuel pumps.

Two sampling stations were chosen on the 

basis of the abundance and diversity of 

organisms available, the different types of 

habitat represented, and their location in the 

stream system. Station 1 was in the middle 

reaches of the stream, where a peak concentration of 11 mg/1 of TFM was maintained for 12 h. 

Passage time for the chemical at this station, 

including the buildup to full strength and 

decline was about 20 h. The samples were taken 

in a 350-m long area consisting of riffles and 

runs where the stream bottom was mostly 

rubble and gravel. Station 2 was about 800 m 

from the mouth of the river and about 10 km (6 

mi) downstream from the last TFM application 

point. The chemical passed this point over a 

period of 20-25 h, and the peak concentration of 

10-11 mg/1 lasted about 7 h. The samples were 

taken in a 100-m long area where the bottom 

consisted mostly of clay overlain by large rubble 

and boulders, with small pockets of sand.

We collected samples at each station at four 

intervals: before treatment; near the end of the 

peak period of concentration of the chemical 

(hereafter called treatment samples); and at 24 

and 96 h after treatment. We assumed that the 

treatment samples would contain the maximum 

residue because they were collected after maximum exposure to the chemical and immediately before exposure to untreated water. Samples 

included water, bottom soil (sand with some

Table 1. Organisms collected in the East Au Gres River, Michigan 

for analysis of residues of TFM

Common Name Classification

Plants

Algae 

Water weed

Invertebrates

Oligochaetes (Aquatic earthworms)

Crayfish

Stonefly

Mayfly

Dragonfly

Dobsonfly

Cranefly

Snipefly

Snails

Fish

Rainbow trout

Cyprinids

Sculpin

Cladophora sp. 

Elodea sp.

Annelida, Oligochaeta 

Crustacea, Decapoda 

Insecta, Plecoptera, Pferonarcys sp. 

Insecta, Ephemeroptera, Ameletus sp. 

Insecta, Odonata, Anisoptera 

Insecta, Megaloptera, Corydalidae 

Insecta, Diptera, Tipulidae 

Insecta, Diptera, Rhagionidae 

Mollusca, Gastropoda, Physa sp.

Sa/mo gqircfner;

Rhinichthys cataractae and Notropis sp.

Cottus sp.
detritus), oligochaetes (aquatic earthworms), 

crayfish, aquatic insect larvae, snails, algae, 

aquatic vascular plants, and fish. Two species 

of cyprinids were pooled as a sample representative of fish which feed at a low trophic level. 

Organisms sampled (Table 1) were those for 

which we could get an adequate weight of 

material for analysis in a reasonable time (2-3 

h). Due to habitat and sampling variations, 

some species were found at only one of the 

stations, and some were not found in all samples 

from the same station.

Water samples were collected in plastic bottles 

at the surface of the stream. Bottom soil samples 

were taken manually from the top 2.5 cm of the 

substrate. Fish, crayfish, and mayflies were 

collected by electrofishing. In the collection of 

other aquatic insects and oligochaetes, one man 

disturbed the bottom material with a shovel 

immediately upstream from a screen held by

another. Snails and aquatic vegetation were 

picked by hand. All samples were frozen shortly 

after collection, in a styrofoam box containing 

dry ice. They remained frozen during transportation to the laboratory for analysis.

Samples were analyzed for TFM residues by 

the method of Alien and Sills (1974), which has a 

detection limit of 0.01 /ug/g. Organisms were 

analyzed on the basis of wet weight, whole-body 

residues. The extraction procedure was modified 

to accommodate the small sample weights of the 

insects, which were homogenized in the hexaneethyl ether (3 + 1) extracting solvent. Each soil 

sample was blended in three, 100-ml quantities 

of solvent. Water samples were acidified and 

extracted with three portions of hexane-ethyl 

ether (3+1). The extracts were cleaned up, 

concentrated, and analyzed by the gas 

chromatographic method of Alien and Sills 

(1974).

RESULTS AND DISCUSSION

No pretreatment samples at either station 

(Tables 2 and 3) showed TFM with the exception 

of oligochaetes, which yielded a small 

chromatographic peak (Table 2) with nearly the 

same retention time as TFM. Treatment 

samples contained the highest concentrations 

of TFM. Posttreatment samples showed a rapid 

decrease in TFM residues with time.

Highest residues were accumulated by 

oligochaetes and snails, the treatment samples 

of which contained 21.4 and 15.3 ^g/gofTFM, 

respectively. No oligochaetes were collected in 

the 24- and 96-h samples. They were not 

abundant even before treatment at station 1 

because the largely gravel and rubble bottom 

there was poor oligochaete habitat. There is, 

however, previous evidence of a significant 

reduction of oligochaete populations after 

treatment of streams with TFM (Torblaa 1968). 

Snails were the only organisms which retained 

more than 0.1 /zg/g of TFM 96 h after treatment; they retained 0.37 /ug/g, a 98% reduction 

from residue levels observed in treatment 

samples.

Fish ranked next in terms of residues present 

immediately after maximum exposure to TFM. 

Residues in fish ranged from 4.3 /ug/g in

rainbow trout to 11.4 /ug/g in cyprinids, both at 

station 2. Fish also retained the highest residues 

at 24 h after treatment. The reduction in 

residues after 24 h ranged from 45 to 87% with 

the exception of rainbow trout at station 2, in 

which the reduction was only 4%. By 96 h after 

treatment, residues had declined 99% in all fish 

samples.

Residues in aquatic insects at the end of 

treatment ranged from 0.8 /ug/g in cranefly 

larvae to 5.73 /ug/g in mayfly nymphs. Concentrations of TFM in insects declined by 85 to 92% 

after 24 h and by 93 to 99% after 96 h in fresh 

water.

Aquatic plants accumulated residues of TFM 

in about the same range as insects. Although 

the number of samples was limited, the data 

indicate that algae (Cladophora spj absorbed 

TFM more rapidly than higher plants (Elodea 

sp.). The concentrations of TFM in plants 

declined by 89 to 97% after 24 h and 98 to 99% 

after 96 h in fresh water.

Concentrations of TFM at the end of treatment were lower in soil samples than in other 

samples, indicating that TFM does not have an 

affinity for soil particles. The maximum concen-
6

tration of TFM in a soil sample was only 0.8 

Atg/g. By 96 h after treatment, no TFM was 

detectable in soil samples.

TFM apparently does not persist in the food 

chain. Sills and Alien (1975) showed that 

residues of TFM in fish muscle decreased to near 

the detection limit by 24 h after exposure to 1 to 4 

mg/1 of TFM in controlled laboratory studies. 

Mechanisms of elimination from fish have been 

outlined by Lech and Costrini (1972) and Hunn 

and Alien (in press). The present study showed

the loss of residues from whole fish under field 

conditions also to be rapid, but somewhat slower 

than under the laboratory conditions of Sills 

and Alien (1975). The difference was likely due 

to the higher concentrations to which fish were 

exposed in the field. Residues of TFM were more 

readily eliminated from aquatic insects than 

from fish. No evidence of biomagnification was 

noted and residues in all major components of 

the stream ecosystem were reduced by 93 to 99% 

in 96 h.

Table 2. TFM residues in samples from the east branch of the 

East Au Gres River (Sample station 1 a ) at State Road

Sample

Water

Soil

Plants (vascular)

Oligochaetes

Crayfish

Stonefly

Dragonfly

Dobsonfly

Cranefly

Snipefly

Rainbow trout

Sculpin

Before 

treatment

0.00

0.00

0.00

0.02

NS

0.00

NS

NS

0.00

0.00

0.00

0.00

TFM residues (

During 

treatment

9.45

0.44

2.90

21.4

NS

2.89

0.85

NS

0.80

1.13

9.80

10.9

! VB/B)

24 h posttreatment

0.00

0.08

0.32

NS

0.33

0.25

0.07

NS

0.06

0.18

1.33

6.00

96 h posttreatment

0.00

0.00

0.08

NS

0.01

0.04

0.04

0.04

0.06

0.04

0.01

0.04

Values less than 0.01 |/g/g are reported as 0.00; NS indicates no sample was collected.

Table 3. TFM residues in samples from lower East Au Gres River (Sample station 2'

Sample

Water

Soil

Algae

Crayfish

Stonefly

Mayfly

Cranefly

Snails

Rainbow trout

Cyprinids

Before

treatment

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

TFM residues (

During

treatment

6.10

0.80

6.75

1.14

4.08

5.73

NS

15.3

4.30

11.4

i MB/B)

24 h posttreatment

0.00

0.14

0.22

0.12

0.38

0.17

NS

0.58

4.15

5.00

96 h posttreatment

0.00

0.00

0.03

0.03

0.06

0.02

0.01

0.37

0.08

0.07

Values less than 0.01 //g/g are reported as 0.00; NS indicates no sample was collected.
REFERENCES

Alien, J. L., and J. B. Sills. 1974. Gas-liquid chromatographic determination of 3-trifluormethyl-4-nitrophenol 

residues in fish. J. Assoc. Off. Anal. Chem. 57(2):387-388.

Applegate, V.C., J. H. Howell, J. W. Moffett, B. G. H. 

Johnson, and M. A. Smith. 1961. Use of 3-trifluormethyl-4-nitrophenol as a selective sea lamprey larvicide. 

Great Lakes Fish. Comm., Tech. Rep. No. 1. 35 pp.

Billy, T. J., S. L. Daniels, L. L. Kempe, and A. M. Beeton. 

1965. Field application of methods for recovery of the 

selective lampricide, 3-trifluoromethyl-4-nitrophenol. 

Univ. Mich., Great Lakes Res. Div., Publ. No. 13:17-24.

Great Lakes Fishery Commission. 1958. Lamprey control 

and research in the United States. Pages 35-50 in Great 

Lakes Fishery Commission annual report for the year 

1958.

Howell, J. H., and W. M. Marquette. 1962. Use of mobile 

bioassay equipment in the chemical control of sea 

lamprey. U.S. Fish Wildl. Serv., Spec. Sci. Rep. Fish. No. 

418. 9 pp.

Hunn, J. B., and J.L. Alien. Some aspects of the dynamics of 

TFM and quinaldine in rainbow trout Salmo gairdneri. 

Comp. Gen. Pharmacol. In press.

Kempe, L. L. 1973. Microbial degradation of the lamprey 

larvicide 3-trifluoromethyl-4-nitrophenol in sedimentwater systems. Great Lakes Fish. Comm., Tech. Rep. No. 

18. 16 pp.

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.

Sills, J. B., and J. L. Alien. 1975. Accumulation and loss of 

residues of 3-trifluoromethyl-4-nitrophenol (TFM) in fish 

muscle tissue: laboratory studies. U.S. Fish Wildl. Serv., 

Invest. Fish Control, No. 65.

Torblaa, R. L. 1968. Effects of lamprey larvicides on 

invertebrates in streams. U.S. Fish Wildl. Serv., Spec. Sci. 

Rep. Fish. No. 572. 13 pp.

(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 spj, 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 

Leif L. Marking and Lee E. Olson. 1975. 27 pp.

61. 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. 1975. 9 pp.

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

and Frog Larvae, by Jack H. Chandler and Leif L. Marking. 1975. 7 pp.

Fish Control Laboratories

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U.S. Department of the Interior

P.O. Box 862

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