INVESTIGATIONS IN FISH CONTROL

69. Toxicity of 3-trifluoromethyl-4-nitrophenol (TFM), 

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

and a 98:2 Mixture to Fingerlings of Seven Fish 

Species and to Eggs and Fry of Coho Salmon

70. The Freshwater Mussel (Anodonta sp.) as an 

Indicator of Environmental Levels of 

3-trifluoromethyl-4-nitrophenol (TFM)

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.

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

69. Toxicity of 3-trifluoromethyl-4-nitrophenol (TFM), 

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

and a 98:2 Mixture to Fingerlings of Seven Fish 

Species and to Eggs and Fry of Coho Salmon

By Terry D. Bills and Leif L. Marking

70. The Freshwater Mussel (Anodonta sp.) as an 

Indicator of Environmental Levels of 

3-trifluoromethyl-4-nitrophenol (TFM)

By Alan W. Maki and Howard E. Johnson

United States Department of the Interior

Fish and Wildlife Service 

Washington, D.C. 1976

TOXICITY OF 3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM),

2f ,5-DICHLORO-4f -NITROSALICYLANILIDE (BAYER 73),

AND A 98:2 MIXTURE TO FINGERLINGS OF SEVEN FISH SPECIES

AND TO EGGS AND FRY OF COHO SALMON

by

Terry D. Bills and Leif L. Marking 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT

We determined the toxicity of the lampricides 3-trifluoromethyl-4-nitrophenol 

(TFM) and 2',5-dichloro-4'-nitrosalicylanilide (Bayer 73) and a 98:2 mixture of these 

compounds against fingerlings of seven species offish brown trout (Salmo trutta), 

rainbow trout (Salmo gairdneri), lake trout (Salvelinus namaycush), brook trout 

(Salvelinus fontinalis), channel catfish (Ictalurus punctatus), bluegill (Lepomis 

macrochirus), and yellow perch (Perca flavescens) and to eggs and fry of coho 

salmon (Oncorhynchus kisutch). Channel catfish were the most sensitive to TFM 

and brown trout to Bayer 73. Bluegills were the most resistant to both TFM and 

Bayer 73. The toxicity of TFM and Bayer 73 individually was influenced far more by 

pH than was the mixture in standard laboratory tests with rainbow trout. Toxicity of 

the mixture was additive or less than additive to all species and life stages tested. The 

mixture was slightly more toxic to larval lampreys (Petromyzon marinus) than to 

other fish in comparable laboratory toxicity tests. The margin of safety was narrow, 

however, when the 24-h toxicity for brown trout or rainbow trout was compared with 

the 24-h LC99 for sea lamprey larvae.

INTRODUCTION

Before 1964, 3-trifluoromethyl-4-nitrophenol 

(TFM) was the only compound used for the 

control of larval sea lampreys (Petromyzon 

marinus) in tributaries of the Great Lakes 

(Applegate et al. 1961). Howell (1964) pointed out 

the ineffectiveness of TFM in water with certain 

physicochemical characteristics. This ineffectiveness prompted the U.S. Fish and Wildlife 

Service to search for compounds that could 

replace TFM or increase the biocidal activity of 

the compound.

Howell et al. (1964) reported that the 

molluscicide 2',5-dichloro-4'-nitrosalicylanilide 

(Bayer 73, also known as Bayluscide®) was 

extremely toxic to larval sea lampreys. 

However, the compound also was highly toxic to 

other fishes (Marking and Hogan 1967). Howell 

et al. (1964) determined that by mixing from 0.5 

to 4% Bayer 73 with TFM, the efficacy was 

increased without loss of the selective toxicity

displayed by TFM. Since use of the TFM:Bayer 

73 (98:2) mixture began in 1964, few, if any, data 

on its safety or toxicity to nontarget organisms 

have been published. Lennon (1967) pointed out 

the need for such data to meet regulatory agency 

requirements for continued use. The history, 

chemistry, and uses of TFM, Bayer 73, and the 

mixture were summarized by Schnick (1972), 

and Hamilton (1974a, 1974b).

The objectives of the present study were to 

determine (1) the toxicity of TFM, Bayer 73, and 

a 98:2 mixture of the compounds against several 

species of fish in soft water; (2) the effects of 

water temperature, hardness, and pH on the 

toxicity of these lampricides to rainbow trout 

(Salmo gairdneri); (3) the toxicity of these 

lampricides to eggs and fry of coho salmon 

(Oncorhynchus kisutch)', and (4) the margin of 

safety for each compound and the mixture for 

nontarget fishes.
METHODS AND MATERIALS

The chemicals used in the toxicity tests were 

field grade TFM (35.7% active ingredient) 

supplied by American Hoechst Chemical Co., 

Somerville, New Jersey, and Bayer 73 (70% 

wettable powder) supplied by Chemagro Corporation, Kansas City, Missouri. Stock 

solutions of both were prepared in water (the 

liquid formulation of TFM measured 

volumetrically and diluted with water). All 

concentrations were based on active ingredients, and the mixture used was 98% 

TFM:2% Bayer 73. To prepare test solutions of 

the desired concentrations, we pipetted portions 

of stock solutions into the test vessels and 

stirred the resulting media to ensure homogeneity.

Fish and coho salmon eggs were obtained 

from National Fish Hatcheries and maintained 

according to procedures outlined by Hunn et al. 

(1968) for fish and by Bills (1974) for fish eggs. In 

addition to eggs and fry of coho salmon, fish 

tested were fingerlings (0.5-1.5 g) of brown 

trout (Salmo trutta), rainbow trout, lake trout 

(Saluelinus namaycush), brook trout 

(Salvelinus fontinalis), channel catfish (Ictalurus punctatus), bluegill (Lepomis 

macrochirus), and yellow perch (Perca 

flavescens). Fish and coho salmon were 

acclimated to test conditions for 24 h preceding 

chemical additions. Procedures outlined by 

Lennon and Walker (1964) were followed for the

toxicity tests with fingerlings, and procedures 

presented by Bills (1974) were followed for tests 

with eggs. Toxicity tests with coho salmon eggs 

and fry were conducted in 2.5-liter glass vessels; 

all other toxicity tests were conducted in 15-liter 

glass vessels.

Test waters of different hardnesses were 

prepared by adding inorganic salts to deionized 

water (Marking 1969). In toxicity tests in which 

the effects of pH were assessed, chemical buffers 

were added to soft water (Marking and Dawson 

1973). The pH's were checked daily and adjusted 

to within ±0.2 pH units. Temperature in the test 

vessels was maintained by immersing the units 

in a water bath.

Data were analyzed to determine LCSO's and 

95% confidence intervals according to methods 

described by Litchfield and Wilcoxon (1949). 

Additive indices and their ranges were 

calculated from the LC50's and 95% confidence 

intervals according to methods described by 

Marking and Dawson (1975). Additive indices 

quantitate the combined activity of the mixture, 

and the confidence intervals define 

significance. Positive index values indicate 

greater than additive toxicity, and negative 

values indicate less than additive toxicity. 

Indices for which confidence intervals overlap 

zero are considered to indicate that the toxicity 

is neither greater nor less than additive.

RESULTS

Channel catfish and brown trout were most 

sensitive to TFM, followed by yellow perch, lake 

trout, rainbow trout, brook trout, and bluegill 

(Table 1). The 96-h LCSO's in soft water at 12 C 

ranged from 0.750 mg/1 for channel catfish to 

4.89 mg/1 for bluegill. The comparative toxicity 

(96-h LC50) of Bayer 73 to the selected species 

ranged from 0.0282 mg/1 for brown trout to 

0.152 mg/1 for bluegill. On the basis of the 

additive index concept, the toxicity of the 

mixture was strictly additive, indicating that 

from a toxicological standpoint there is no 

advantage in applying the compounds as a 

mixture.

In tests to determine the effects of water 

temperature, hardness, and pH on toxicity of the 

lampricides to rainbow trout, water 

temperatures influenced toxicity least (Table 2). 

TFM and Bayer 73 individually were slightly 

more toxic to rainbow trout in soft water at 17 C 

than at lower temperatures, but the mixture was 

not affected by temperature.

The effect of water hardness on the toxicity of 

the lampricides to rainbow trout was determined in tests with waters of differing 

hardnesses: very soft (10 mg/1 as CaCO3), soft 

(44 mg/1), hard (160 mg/1), and very hard
(300 mg/1). The toxicity of TFM was about 1.5 

times greater in very soft water than in soft, 

hard, or very hard water (Table 2). In contrast, 

the toxicity of Bayer 73 was not affected by 

water hardness. The toxicity of the mixture was 

strictly additive. However, separate evaluation 

of the components of the mixture showed that 

the 96-h LCSO's for the Bayer 73 component were 

not significantly different from the 96-h LCSO's 

for Bayer 73 singly, but that the amount of TFM 

required to produce the same effect was reduced 

twofold to threefold. This difference indicates 

that, it could be economically advantageous to 

apply the mixture if the response of sea 

lampreys is similar to that of rainbow trout and 

if the selectivity of TFM toward the sea lamprey 

could be maintained.

Of the water quality characteristics examined, pH had the most distinguishable effect 

(Table 2). In producing toxicosis to rainbow 

trout in soft water, TFM was 4% as effective at 

pH 9.5 (96-h LC50, 25.2 mg/1) as at pH 6.5 

(0.949 mg/1). The toxicity of Bayer 73 showed a 

similar trend; in producing toxicosis in soft

water, this chemical was 14.3% as effective at 

pH 9.5 (96-h LC50, 0.185 mg/1) as at pH 6.5 

(0.0261 mg/1). As in the other tests, the toxicity 

of the mixture was additive or less than 

additive. The individual evaluation of the 

components showed that the 96-h LCSO's for 

Bayer 73 were similar to those of the compound 

individually, whereas the LC50's for TFM were 

reduced by more than 50% in soft water of pH 

9.5. This relation suggests that most of the 

toxicosis produced by the mixture is attributable 

to the Bayer 73 component in water at pH 9.5.

Among life stages of coho salmon tested, 

green eggs were most sensitive to TFM, followed 

by fry, sac fry, and eyed eggs; the 96-h LC50's 

ranged from 0.639 mg/1 for green eggs to 

3.49 mg/1 for eyed eggs (Table 3). The toxicity of 

Bayer 73 to eggs and fry was dissimilar to that 

of TFM; sac fry and fry were most sensitive, and 

green eggs and eyed eggs were more resistant. 

The 96-h LCSO's ranged from 0.066 mg/1 for sac 

fry to 0.509 mg/1 for eyed eggs. The additive 

index shows that the toxicity of the mixture is 

additive.

Table 1. Toxicity and additive indices of TFM:Bayer 73 (98:2), based on active ingredient, to

seven species of fish in soft water at 12 C.

Species

and 

toxicant

96-h LC50 and 95% 

confidence interval (mg/1)

Individually Combination

Additive 

index

Brown trout

TFM

Bayer 73

Rainbow trout

TFM

Bayer 73

0.940 

0.791-1.17

0.0282 

0.0219-0.0363

1.81 

1.53-2.14

0.0346 

0.0297-0.0404

0.980 

0.810-1.19

0.0200 

0.0165-0.0242

1.16 

0.998-1.35

0.0237 

0.0204-0.0275

-0.752 

-1.61 to -0.147

-0.326 

-0.808 to +0.0295
Table 1. Toxicity and additive indices of TFM:Bayer 73 (98:2), based on active ingredient, to 

seven species of fish in soft water at 12 C (Con't).

Species

and 

toxicant

96-h LC50 and 95% 

confidence interval (mg/l)

Individually Combination

Additive 

index

Lake trout

TFM

Bayer 73

Brook trout

TFM

Bayer 73

Channel catfish

TFM

Bayer 73

Bluegill

TFM

Bayer 73

Yellow perch

TFM

Bayer 73

1.78 

1.51-2.10

0.0490 

0.0433-0.0555

1.83 

1.35-2.48

0.0470 

0.0364-0.0607

0.750 

0.621-0.906

0.0370 

0.0298-0.0459

4.89 

4.27-5.60

0.152 

0.135-0.172

1.71 

1.47-1.98

0.0639 

0.0568-0.0726

1.39 

1.12-1.72

0.0284 

0.0229-0.0351

1.16 

0.786-1.71

0.0237 

0.0160-0.0349

0.615 

0.542-0.697

0.0125 

0.0111-0.0142

3.18 

2.57-3.94

0.0636 

0.0514-0.0788

0.900 

0.723-1.12

0.0184 

0.0148-0.0229

-0.360 

-0.950 to +0.0571

-0.138 

-1.23 to +0.723

-0.158 

-0.599 to +0.190

-0.0687 

-0.506 to +0.320

+0.228 

-0.165 to +0.757
Table 2. Toxicity and additive indices of TFM:Bayer 73 (98:2) based on active ingredients to 

rainbow trout in laboratory tests at selected temperatures, water hardnesses, and pH's.

Temp. Water 

(C) hardness P

7 Soft 7.5

96-h LC50 and 95%

_ . confidence interval (mg/l) Toxicant

Individually

TFM 2.13

1.73-2.62

Bayer 73 0.0620

0.0568-0.0677

12 Soft 7.5

17 Soft 7.5

TFM 1.81

1.53-2.14

Bayer 73 0.0346

0.0297-0.0404

TFM 1.74

1.33-2.27

Bayer 73 0.0439

0.0396-0.0487

12 Very soft 8.1

TFM 9.00

8.13-9.96

Bayer 73 0.0800

0.0650-0.0984

12 Soft 8.1

TFM 14.1

11.4-17.4

Bayer 73 0.0755

0.0649-0.0878

12 Hard 8.1

TFM 14.1

11.4-17.4

Bayer 73 0.100

0.0817-0.122

Combination

1.42

1.15-1.76

0.0290

0.0234-0.0358

1.16

0.998-1.35

0.0237

0.0204-0.0275

1.41

1.14-1.74

0.0288

0.0232-0.0366

3.89

3.18-4.76

0.0794

0.0649-0.0970

3.62

3.16-4.14

0.0738

0.0646-0.0845

4.85

4.03-5.83

0.0989

0.0823-0.119

Additive 

index

-0.134

-0.648 to +0.275

-0.326

-0.808 to +0.0295

-0.466

-1.21 to +0.0219

-0.425

-1.08 to +0.0216

-0.234

-0.665 to +0.0901

-0.333

-0.968 to +0.104
Table 2. Toxicity and additive indices of TFM:Bayer 73 (98:2) based on active ingredients to 

rainbow trout in laboratory tests at selected temperatures, water hardnesses, and pH's (Con't).

Temp. Water 

(C) hardness

12 Very hard 8.1

96-h LC50 and 95%

_ . confidence interval (mg/l) Toxicant

Individually

TFM 17.3

14.0-21.4

Bayer 73 0.0865

0.0756-0.0990

12 Soft 6.5

TFM 0.949

0.840-1.07

Bayer 73 0.0261

0.0197-0.0345

12 Soft 8.5

TFM 5.40

4.58-6.37

Bayer 73 0.0705

0.0552-0.0901

12 Soft 9.5

TFM 25.2

20.4-31.1

Bayer 73 0.185

0.133-0.257

Combination

4.65

3.90-5.54

0.0949

0.0796-0.113

0.750

0.669-0.841

0.0153

0.0136-0.0172

3.70

3.09-4.43

0.0755

0.0630-0.0898

9.30

8.20-10.5

0.190

0.167-0.215

Additive 

index

-0.366

-0.890 to +0.0139

-0.377

-0.871 to -0.0194

*' u.V>.

-0.756

-1.60 to -0.181

-0.396

-1.13 to +0.0947
Table 3. Toxicity and additive indices of TFM:Bayer 73 (98:2), based on active ingredient, to

eggs and fry of coho salmon in soft water at 12 C.

Stage of 

development 

and toxicant

96-h LC50 and 95% 

confidence interval (mg/l)

individually Combination

Additive 

index

Green eggs

TFM

Bayer 73

Eyed eggs

TFM

Bayer 73

Sac fry

TFM

Bayer 73

Swim-up fry

TFM

Bayer 73

0.639 

0.461-0.886

0.286 

0.212-0.387

3.68 

3.17-4.27

0.509 

0.369-0.703

3.49 

3.14-3.88

0.0655 

0.0582-0.0737

2.39 

2.12-2.69

0.0679 

0.0582-0.0792

0.860 

0.609-1.21

0.0175 

0.0124-0.0247

2.59 

1.97-3.41

0.0528 

0.0402-0.0696

1.41 

1.13-1.75

0.0282 

0.0227-0.0350

1.89 

1.57-2.28

0.0386 

0.0320-0.0465

-0.407 

-1.74 to +0.390

+0.238 

-0.264 to +0.928

+0.198 

-0.159 to +0.669

-0.359 

-0.874 to +0.0125
DISCUSSION

Howell et al. (1964) first recognized that the 

effectiveness of TFM was reduced in hard, 

alkaline waters. This phenomenon was later 

quantitated by Marking and Olson (1975) and 

Dawson et al. (1975). Marking and Olson 

reported a 59-fold decrease in the toxicity of 

TFM to lake trout as pH increased from 6.5 to 

9.5, and Dawson et al. (in press) reported an 8- 

fold decrease in the toxicity of TFM to sea 

lamprey ammocetes as the pH increased from 

6.5 to 8.5. As the pH shifts to the alkaline range, 

a concomitant reduction in the availability of 

the free phenol form of TFM decreases the 

amount available to produce toxicosis. Hunn 

and Alien (1974) pointed out that the decrease in 

toxicity at higher pH's is probably caused by the 

reduction in concentration of the lipid-soluble 

free phenol form. Our data for TFM individually 

show the same results about a 25-fold decrease 

in the toxicity of TFM to rainbow trout between 

pH's 6.5 and 9.5.

Howell et al. (1964) killed all sea lamprey 

ammocetes with a mixture of TFM and Bayer 73

at concentrations which were nontoxic when 

the compounds were applied singly. They 

interpreted this as synergistic activity. Dawson 

et al. (in press), using the additive index concept, 

determined that the toxicity of the mixture to 

ammocetes was additive or less than additive 

not synergistic. Data from our study with fish 

also show less than additive or strictly additive 

toxicity. Howell et al. (1964) pointed out the 

economic advantage of applying the mixture; 

i.e., the reduction in the amount of TFM required 

to produce toxicosis without loss of selectivity. 

The reduction is especially significant in the 

treatment of alkaline waters, when use of the 

mixture reduces the amount of TFM needed by 

as much as 50%. However, 24-h TFM toxicity 

data for rainbow trout and brown trout from the 

present study and comparable laboratory data 

for lamprey larvae (Dawson et al. in press) show 

that the margin of safety to nontarget fish is 

reduced when the mixture is applied. A 38% 

mortality of rainbow trout and 10% mortality of 

brown trout can be expected during a treatment 

that kills 99% of the ammocetes.

CONCLUSIONS

1. Of the species tested, channel catfish proved 

to be the most sensitive to TFM; the 96-h 

LC50 was 0.750 mg/1.

2. Brown trout were the most sensitive 

salmonid tested, and bluegills were the most 

resistant to both TFM and Bayer 73.

3. The toxicity of TFM or Bayer 73 was 

influenced little by water hardness; both were 

slightly more toxic at relatively high 

temperatures, and both were significantly 

more toxic at low than at high pH's; as pH 

increased from 6.5 to 9.5, toxicity of TFM 

decreased by a factor of 25 and Bayer 73 by a 

factor of 7.

4. Among early life stages of coho salmon, 

green eggs were most sensitive to TFM, 

followed by fry, sac fry, and eyed eggs. Bayer 

73 was most toxic to sac fry, followed by fry, 

green eggs, and eyed eggs.

5. The mixture of TFM:Bayer 73 was additive or 

less than additive (not synergistic) in toxicity 

to fish under various test conditions.

6. Toxicity of the mixture was influenced less 

than that of the individual toxicants by 

temperature, water hardness, and pH.

7. The mixture was more toxic to larval sea 

lampreys than to nontarget fish in comparable laboratory toxicity tests. However, 

the margin of safety based on the 24-h 

toxicity data with brown trout or rainbow 

trout and the 24-h LC99 with sea lamprey 

larvae was narrow.
LITERATURE CITED

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.

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

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

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

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 No. 63. 13 pp.

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

Laboratory efficacy of 3-trifluoromethyl-4-nitrophenol 

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

a 98:2 mixture as lampricides. U.S. Fish Wildl. Serv., 

Invest. Fish Control, (in press).

Hamilton, S. E. 1974a. A review of the literature on the 

use of Bayluscide in fisheries. FWS [Fish Wildl. Serv.] 

LR [Lit. Rev.] 74-02. No. PB-235 441/AS. National 

Technical Information Service, Springfield, Va. 60 pp.

Hamilton, S. E. 1974b. A review of the literature on the 

use of TFM:Bayluscide in fisheries. FWS [Fish Wildl. 

Serv.] LR [Lit. Rev.] 74-03. No. PB-237 

442/AS. National Technical Information Service, 

Springfield, Va. 39 pp.

Howell, J. H., E. L. King, Jr., A. J. Smith, and L. H. 

Hanson. 1964. Synergism of 5,2'-dichloro-4'-nitrosalicylanilide and 3-trifluormethyl-4-nitrophenol in a 

selective lampricide. Great Lakes Fish. Comm., Tech. 

Rep. No. 8. 21 pp.

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.

Hunn, J. B., and J. A. Alien. 1974. Movement of drugs 

across the gills of fishes. Annu. Rev. Pharmacol. 

14:47-55.

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 No. 1 (Circ. No. 185). 15 

pp.

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

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

Marking, L. L. 1969. Toxicological assays with 

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

Marking, L. L. 1973. Toxicity of quinaldine sulfate to 

fish. U.S. Fish Wildl. Serv., Invest. Fish Control No. 

48. 8 pp.

Marking, L. L., and J. W. Hogan. 1967. Toxicity of Bayer 

73 to fish. U.S. Fish Wildl. Serv., Invest. Fish Control No. 

19. 10 pp.

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

assessment of toxicity or efficacy of mixtures of 

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

No. 67. 8 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. 27 pp.

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 No. 

44. 31 pp.

THE FRESHWATER MUSSEL (ANODONTA SP.)

AS AN INDICATOR OF ENVIRONMENTAL LEVELS OF

3-TRIFLUOROMETHYL-4-NITROPHENOL(TFM) 1

by

Alan W. Maki2 and Howard E. Johnson 

Department of Fisheries and Wildlife,

Michigan State University 

East Lansing, Michigan 48824

ABSTRACT

After freshwater mussels (Anodonta sp.) were exposed to 8.68-mg/l solutions of 

3-trifluoromethyl-4-nitrophenol (TFM; 14C-TFM and analytical grade TFM) in a 

model stream for 24 h, uptake and elimination rates of TFM residues for three body 

components were determined by radioassay. The average residue concentrations 

(jigTFM/g wet wt) after the 24-h exposure were 44.4 in the foot, 37.7 in the gill, and 

38.5 in the viscera. The average calculated half-time for residue elimination from 

the three components was 20.2 h. The rate of uptake and ultimate residue 

concentration was widely variable, presumably because the feeding and locomotor 

activity of individual mussels varied greatly during the exposure period.

INTRODUCTION

The use of benthic invertebrates as indicators 

of water quality has long been a useful 

procedure in pollution investigations (American 

Public Health Association 1971). Among 

bivalve mollusks used for monitoring insecticides, oysters have been used in the marine 

environment (Bugg et al. 1967; Casper 1967) and 

several species of mussels (Unionidae) in fresh

Michigan Agricultural Experiment Station Journal Article 

No. 7042. The research was supported by U.S. Fish and 

Wildlife Service Contract 14-16-0008-651.

2Present address: Environmental Water Quality Research 

Department, The Procter & Gamble Company, Ivorydale 

Technical Center, Cincinnati, Ohio 45217.

waters (Bedford et al. 1968; Miller et al. 1966). 

The filter feeding habit of freshwater mussels 

results in the accumulation of many elements in 

their tissues from water, against concentration 

gradients (Gaglione and Ravera 1964). The 

sedentary nature of the mussel makes it an ideal 

candidate for pesticide monitoring because the 

animal cannot escape a toxicant by drifting or 

swimming away.

We report here the results of experiments 

designed to evaluate the freshwater mussel 

(Anodonta sp.) as an indicator of residues of the 

larval lampricide 3-trifluoromethyl-4- 

nitrophenol (TFM) after a simulated treatment 

for lamprey control in a model stream system.
METHODS AND MATERIALS

Mussels of the genus Anodonta, 8 to 10 cm 

long, were collected from the Muskegon River at 

Evart, Michigan. Inasmuch as species identification could not be confirmed without 

sacrificing the animals, all individuals were 

simultaneously collected from the same pool 

and identified to the genus Anodonta from 

external characters. The mussels were maintained in hatchery channels at the State Fish 

Hatchery in Paris, Michigan, at water 

temperatures of 10 to 12 C.

The rate of uptake and elimination of 14CTFM by the mussels was tested in a model 

stream system inside the hatchery in fall 1973. 

The stream consisted of a concrete trough 4.0 m 

long and 0.6 m wide which drained into a 

second trough of the same dimensions. Water 

was supplied to the model stream at 100 1/min 

from Cheney Creek, a small natural stream 

adjacent to the hatchery. In the upper trough, 

which was designated as a pool, the water depth 

was maintained at 25 cm. Gravel and rubble 

were transferred from a nearby stream to the 

lower trough, which was designated as a riffle 

section. Drifting organisms and organic matter 

from Cheney Creek settled in the troughs over a 

period of about 1 yr before the tests were 

conducted. Overhead fluorescent lamps 

(Coolwhite) provided light intensity of 

9688 ± 538 Ix at the stream surface. Photoperiod was controlled to conform to natural 

daylength, with seasonal adjustments.

One month before the experimental treatment, 50 mussels were placed in the experimental pool of the model stream for acclimation. On 

13 November 1973, water flow to the troughs 

was stopped and the drains were plugged. Water 

was then recirculated by placing a large 

diaphragm pump in the downstream end of the 

stream, with the discharge at the head of the 

channel. The pump capacity was slightly more 

than 100 1/min, which caused the introduction 

of some air into the pump and facilitated reaeration of the stream water. The stream 

volume was 500 to 525 liters. The entire model 

stream was treated with an isotope dilution of 

about 1.0 mCi of 14C-TFM and 4.500 g of 

analytical grade TFM, all in 10 ml of acetone. 

This dilution gave approximately 2.6 x 106

counts/min/1 at time zero and an actual 

concentration of 8.68 mg/1 labeled and unlabeled TFM in the stream water. Water 

temperature was 11 C, pH 7.8, and hardness 211 

mg/1 as CaCO3 .

After the 24-h exposure period, the discharge 

from the pump was directed into a 980-liter 

metal container and a flow of fresh water was 

immediately reestablished in the model stream. 

The 14C-TFM was then recovered by acidifying 

the water to pH 4.0 and passing it through a 

column of non-ionic polymeric adsorbent (Lech 

1971).

Two mussels were removed at intervals of 1, 

2.5, 10, 14, and 24 h, thoroughly rinsed in clean 

water, placed in labeled bags, and immediately 

frozen. Additional samples were removed at 

intervals up to 30 days after exposure for 

determination of elimination rates. The samples 

were kept frozen for about 1 wk before analysis. 

The mussells were dissected from their shells 

and separated into three components: foot, 

gills, and viscera. Four replicate analyses were 

conducted for each component. Each sample 

was dried at 50 C for 48 h, and its weight 

adjusted to 100 to 150 mg. Samples were 

combusted to 14CO2 and water in a semiautomated Nuclear Chicago combustion apparatus. The 14CO2 was taken up in 10 ml of 

monoethanolamine-methyl cellosolve, 1:2 

(V/V). A 2-ml portion was then radioassayed in 

15 ml of a mixture of toluene-methyl cellosolve, 

2:1 (V/V), and fluor with a dual channel Nuclear 

Chicago Unilux I (Model 6850) liquid scintillation spectrometer. At each sampling interval 

2-ml aliquots of the water samples were 

radioassayed in 15 ml of a toluene-trition X-100 

fluor, 2:1 (V/V).

We established efficiency "curves for the 

instrument by using a series of internally 

quenched standards, and converted all sample 

counts to actual disintegrations per minute and 

//Ci values, using the channel ratio and 

efficiency curve. The isotope dilution factor was 

the basis for calculation of actual residue 

concentrations on a wet and dry weight basis for 

all samples.
RESULTS AND DISCUSSION

All individual mussels concentrated residues 

of TFM by about 3 to 4 times over the ambient 

water concentration during the 24-h exposure 

(Table 1). However, significant variations 

existed between individuals collected within 

each sampling period. The amount of locomotor 

activity and length of time the shell is open with 

foot extended apparently has a direct bearing on 

the bioconcentration of TFM residues by the soft 

internal portions of the mussel. During the 

exposure, mussels were observed in all stages of 

locomotion, ranging from foot extended to a 

completely nonmotile state with a closed shell. 

These individual variations in behavior 

probably explain the wide variation in uptake 

observed among the individual mussels.

The data were further characterized by the use 

of a simple linear regression of //g TFM/g 

tissue on a dry weight basis against exposure 

time in hours. The equation was of the general

form:

where Y = concentration of total TFM residue 

expressed as pg/g dry weight, a = the Yintercept of regression, b = rate of loss or slope 

of the regression, and X = exposure time in 

hours.

The regression intercept, regression coefficient, sample standard deviation of the regression coefficient S^, and confidence intervals of 

the slope were calculated according to Steele 

and Torrie (1960). The calculated equations for 

foot, gill, and visceral fractions demonstrated 

the relatively rapid uptake rates after initial 

exposure; the slopes were 10.5, 7.8, and 9.0, 

respectively (Table 2).

The mussels eliminated most TFM residues 

within 24 h after exposure but detectable 

residues were present in most samples taken as 

long as 4 wk after exposure (Table 3). Neither the 

TFM residue concentrations nor the elimination 

rates differed significantly among the foot, gill, 

and viscera fraction. The same wide variation

Table 1. Average concentration ( ± one standard deviation in parentheses) of TFM residues 

(fi g TFM/g tissue) in the foot, gills, and viscera of mussels (Anodonfasp.) after exposure 

to 8.68 mg/l solutions of TFM for the indicated periods. Each value is the mean of four 

samples from two mussels.

Body 

component

and type of 

weight ( j*g/g) 1

Exposure time (hours)

2.5 10 14 14

Foot

Dry 

Wet

Gill

Dry 

Wet

Viscera

Dry 

Wet

18.1 (19.2)

3.2 (3.4)

34.9 (36.1)

6.4 (6.8)

18.4 (20.3)

1.9 (3.1)

79.3 (13.2)

11.8 (1.6)

93.9 (37.8)

9.3 (1.1)

96.6 (32.0)

15.8 (3.5)

66.4 (17.9)

12.4 (6.0)

98.2 (41.5)

13.0 (7.0)

78.1 (23.5)

11.2 (4.9)

209.2 (110.6)

36.6 (27.7)

168.9 (48.1)

32.5 (11.7)

185.4 (43.8)

26.7 (8.1)

268.8 (103.2) 

44.4 (37.7)

232.3 (97.5) 

37.7 (9.3)

248.4 (106.2) 

38.5 (11.3)
Table 2. Regression equations describing relation between concentrations of TFM (Y) and 

time (X) for the foot, gill, and viscera portions of mussels (Anodonta sp.) with 95% 

confidence intervals of the slope.

Body component

and stage 

of experiment

Regression equation

95% 

Confidence intervals

Foot

Uptake 

Elimination

Gill

Y = 20.6 + 10.5 (X) 

Y = 30.9 -11.8 (log X)

7.9

0.05

Uptake

Elimination

Viscera

Uptake

Elimination

y =

y =

y =

y =

45.

40.

37.

32,

.3 +

.2 -

.6 +

.6 -

7.8

15.4

9.0

12.5

(X)

(log

(X)

(log

X)

X)

4.

0.

6.

0.

4

.06

9

,05

among individuals observed in the uptake rates 

was apparent during the elimination period.

The rate of TFM elimination was described by 

a regression of actual concentrations of TFM 

determined from radioassay of each body 

component against the log time in hours. The 

data are described by the following general 

equation:

Y = a + (-b) (log X)

where Y = concentration of total TFM residue 

in the organism expressed as // g/g dry weight, 

a = the Y-intercept of regression or initial 

concentration in tissue at the initiation of the 

elimination period, b - rate of loss or slope of 

the regression, and log X - log of time in hours.

The calculated data for elimination of TFM 

from each of the mussel body components 

indicate that the half-lives of TFM residue 

concentrations were 20.4, 20.2, and 20.1 h for 

foot, gill, and visceral components, respectively 

(Table 2). More rapid elimination rates were 

determined from mussels collected after an 

actual TFM treatment of the Ocqueoc River. The 

mussels sampled had eliminated 96% of their 

body lampricide residues within 24 h and more 

than 99% within 96 h (J. L. Alien and J. B. Sills, 

unpublished data). These more rapid elimination rates may be due to the much higher flow 

rate and water volume of the Ocqueoc River, 

which diluted the residue more rapidly than it 

was diluted in our model stream.
Table 3. Average concentration ( ± one standard deviation in parentheses) of TFM residues 

( y. g TFM/g tissue) in the foot, gills, and viscera of mussels (Anodonta sp.) at indicated 

times after a 24-h exposure to 8.68 mg/l TFM.

Withdrawal

period

(h)

7

9

12

20

34

57

300

325

710

Foot

Wet

weight

1.4 (0.5)

8.5 (1.0)

0.8 (0.5)

6.5 (7.1)

0.8 (0.3)

0.4 (0.3)

0.2 (0.1)

0.1 (0.1)

0.0 (- -)

Dry

weight

7.1 (1.7)

39.4 (3.7)

3.4 (2.3)

39.4 (43.3)

4.3 (1.7)

2.1 (1.6)

1.1 (0.8)

0.5 (0.9)

0.2 (0.3)

Gill

Wet

weight

1.9 (0.5)

7.7 (1.1)

0.8 (0.2)

5.4 (5.9)

1.2 (0.4)

0.2 (0.3)

0.2 (0.1)

0.1 (0.1)

0.1 (0.6)

Dry

weight

12.0 (2.9)

55.0 (6.4)

5.4 (1.0)

36.7 (39.4)

11.6 (2.8)

1.5 (2.7)

1.3 (0.4)

0.8 (0.7)

0.7 (0.8)

Viscera

Wet

weight

1.5 (0.1)

6.6 (1.2)

0.8 (0.2)

5.7 (6.0)

1.0 (0.5)

0.1 (0.2)

0.1 (0.1)

0.2 (0.1)

0.0 (- -)

Dry

weight

8.2 (1.0)

39.0 (9.2)

5.6 (0.8)

40.7 (42.8)

6.1 (3.6)

1.0 (1.2)

0.4 (0.5)

1.3 (0.4)

0.0 (--)

CONCLUSIONS

The mussel Anodonta sp. can concentrate 

TFM to a level of 3 to 4 times the ambient water 

concentration during a 24-h exposure. TFM is 

rapidly eliminated after exposure ceases; more 

than half the residue is lost within 24 h. Total 

residue concentrations vary widely among 

individual mussels, probably because of individual variations in activity during the period 

of exposure. Although Anodonta sp. may be a 

useful indicator of recent TFM contamination, 

the variable rate of uptake among individual 

organisms limits its value as a quantitative 

method for monitoring TFM concentrations.

LITERATURE CITED

American Public Health Association. 1971. Standard 

methods for the examination of water and 

wastewater. 13th ed. 874 pp.

Bedford, J. W., E. W. Roelofs, and M. J. Zabik. 1968. The 

freshwater mussel as a biological monitor of pesticide 

concentrations in a lotic environment. Limnol. 

Oceanogr. 13:118-126.

Bugg, J. C., J. E. Higgins, and E. A. Robertson. 1967. Chlorinated pesticide levels in the eastern 

oyster (Crassostrea uirginica) from selected areas of the 

South Atlantic and Gulf of Mexico. Pestic. Monit. J. 1: 

9-12.

Casper, V. L. 1967. Galveston Bay pesticide study 

water and oyster samples analyzed for pesticide residues 

following mosquito control program. Pestic. Monit. J. 1: 

13-15.

Gaglione, P., and O. Ravera. 1964. Manganese 54 concentration in fall-out water, and Unio mussels of Lake 

Maggiore, 1960-1963. Nature 204:1215-1216.

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

nitrophenol (TFM) in the rat. Toxicol. Appl. Pharmacol. 

20:216-226.

Maki, A. W. 1974. The effects and fate of larval lampricide (TFM: 3-trifluoromethyl-4-nitrophenol) in model 

stream communities. Ph.D. Thesis, Michigan State 

Univ., East Lansing. 162 pp.

Miller, C. W., B. M. Zuckerman, and A. J. Charig. 

1966. Water translocation of diazinon-14C and 

parathion-35S off a model cranberry bog and subsequent 

occurrence in fish and mussels. Trans. Am. Fish. Soc. 

95:345-349.

Steele, R. G. D., and J. H. Torrie. 1960. Principles and 

procedures of statistics. McGraw-Hill Book Co., Inc. 

481 pp.



(Reports 53 through 55 are in one cover.)

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

Marking. 1973. 11 pp.

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

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

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

Sulfate:MS-222, by Joe B. Sills, John L. Alien, Paul D. Harman, and Charles W. Luhning. 1973. 

12pp.

(Reports 56 through 59 are in one cover.)

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

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

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

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

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

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

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

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

(Reports 60 through 62 are in one cover.)

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

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

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

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

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

(Reports 63 through 66 are in one cover.)

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

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

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

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

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

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

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

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

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

V. K. Dawson. 1975. 7 pp.

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

Ralph M. Burress. 1975. 8 pp.

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