INVESTIGATIONS IN FISH CONTROL

56. Toxicity of the Lampricide 3-Trifluoromethyl-4- 

nitrophenol (TFM) to 1O Species of Algae

57. Acute Toxicities of 3-Trifluoromethyl-4-nitrophenol

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

to Larvae of the Midge Chironomus tentans

58. Acute Toxicity of the Lampricide 3-Trifluoromethyl-4- 

nitrophenol (TFM) to Nymphs of Mayflies (Hexagenia sp.)

59. Toxicity and Residue Dynamics of the Lampricide 

3-Trifluoromethyl-4-nitrophenol (TFM) in 

Aquatic Invertebrates

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 45-46 (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 Application 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.

Continued on inside back cover 
INVESTIGATIONS IN FISH CONTROL

56. Toxicity of the Lampricide 3-Trifluoromethyl-4- 

nitrophenol (TFM) to 10 Species of Algae

By Alan W. Maki, Lester D. Geissel, and 

Howard E. Johnson

57. Acute Toxicities of 3-Trifluoromethyl-4-nitrophenol 

(TFM) and 2% 5-Dichloro-4f -nitrosalicylanilide 

(Bayer 73) to Larvae of the Midge Chironomus tentans

By Joseph A. Kawatski, Margaret M. Ledvina, 

and Carl R. Hansen, Jr.

58. Acute Toxicity of the Lampricide 3-Trifluoromethyl-4~ 

nitrophenol (TFM) to Nymphs of Mayflies (Hexagenia sp.)

By Calvin R. Fremling

59. Toxicity and Residue Dynamics of the Lampricide 

3-Trifluoromethyl-4-nitrophenol (TFM) in Aquatic 

Invertebrates

By Herman O. Sanders and David F. Walsh

United States Department of the Interior

Fish and Wildlife Service

Washington, D,C. 1975
IN THIS COVER

Pages

Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol 

(TFM) to 10 Species of Algae, by Alan W. Maki, Lester D. 

 Geissel, and Howard E. Johnson. .................... 1-17

Acute Toxicities of 3-Trifluoromethyl-4-nitrophenol (TFM) 

and 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) to 

Larvae of the Midge Chironomus tentans, by Joseph A. 

Kawatski, Margaret M. Ledvina, and Carl R. Hansen, Jr. ... 1-7

Acute Toxicity of the Lampricide 3-Trifluoromethyl-4- 

nitrophenol (TFM) to Nymphs of Mayflies (Hexagenia sp.), 

by Calvin R. Fremling ............. ........ ...... 1-7

Toxicity and Residue Dynamics of the Lampricide 

3-Trifluoromethyl-4-nitrophenol (TFM) in Aquatic 

Invertebrates, by Herman O. Sanders and David F. Walsh. . . . 1-9
FOREWORD

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

Great Lakes. While the toxicity of TFM to lamprey is well documented (Investigations in Fish Control, number 44), its effects and those of mixtures 

of TFM with Bayer 73 (2-aminoethanol salt of 2'5-dichloro-4f -nitrosalicylanilide) on other organisms are unknown. The toxicity of Bayer 73 to fish 

was reported in IFC, number 19.

A petition for the registration of TFM must include data describing its 

effects on the non-target biota and on its fate within treated animals and 

in the environment. Studies were conducted at the Fish Control Laboratory, 

La Crosse, Wisconsin and under contract by researchers at other laboratories to develop the necessary data.

The following papers concern the effects of TFM on selected species of 

algae and of TFM, Bayer 73, and/or mixtures of the two on selected invertebrates under laboratory conditions. These papers represent the first 

of a continuing series to be published in Investigations In Fish Control. 

All will be used to support petitions for registration to permit the continued use of lampricides. Subsequent papers in the IFC series will concern the toxicity of TFM to fishes, invertebrates, and macrophytes, the 

efficacy of various TFM formulations, the residue patterns associated 

with applications, and the biotransformation of TFM in fish and invertebrates.

Fred P. Meyer, Director 

Fish Control Laboratories

iii

INVESTIGATIONS IN FISH CONTROL

56. Toxicity of the Lampricide 3-Trif luoromethyl-4- 

nitrophenol (TFM) to 1O Species of Algae

By Alan W. Maki, Lester D. Geissel, 

and Howard E. Johnson

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. 1975
CONTENTS

Page 

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

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

Methods ...................................... 3

Results. ...................................... 7

Toxicity tests ............................... 7

Specific growth rates .......................... 7

Regression analysis of toxicant concentration and 

maximum standing crop ....................... 7

Cell counts ................................. 8

Mortality tests. .............................. 10

Discussion .................................... 15

Conclusions. ................................... 16

Acknowledgments. ............................... 16

References .................................... 17
TOXICITY OF THE LAMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL

(TFM) TO 10 SPECIES OF ALGAE

By Alan W. Maki, Lester D. Geissel, and Howard E. Johnson

Department of Fisheries and Wildlife 

Pesticide Research Center, Michigan State University 

East Lansing, Michigan

ABSTRACT

The toxicity of analytical and field grades of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to unialgal cultures of four green algae, four 

blue-green algae, and two species of diatoms was examined in 96-h toxicity tests. Growth was measured by daily optical density readings, cell 

counts of nonfilamentous species, and a gravimetric determination of 

maximum standing crop at the end of the tests. A 50% inhibition of growth 

(calculated from optical density data) occurred at concentrations less 

than 10 mg/1 of TFM for all species tested except Chlorella pyrenoidosa, 

which was resistant at concentrations greater than 15 mg/1. Inhibition of 

growth was lowest in the blue-green species (50% effect levels, 9-10 

mg/1), generally intermediate in the green algae, and severe in diatoms 

(50% effect levels, 1-4 mg/1). Field grade TFM tended to be more toxic 

than the analytical grade. Similar toxicity relationships were indicated by 

each of the three types of measurements made.

Growth tests conducted with high concentrations of TFM and subsequent 

filtration of the cells and resuspension in toxicant-free medium indicated 

that exposure to TFM at concentrations of 30 mg/1 for 96 h did not destroy the viability of algal cells but temporarily inhibited growth.

INTRODUCTION

The toxicity of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to representatives of most animal groups has been examined 

in several investigations (see Schnick 1972 

for a review), but knowledge of the effects of 

the toxicant on nontarget plant and algal 

species is limited (Howell 1966; Haas 1970). 

The compound is known to be phytotoxic and 

has been used at relatively high concentrations for the control of rooted aquatic plants 

(Josephs 1961). It is necessary to determine 

the effects of TFM on a variety of algal species, for it is well understood that any 

changes in these primary producers will influence each successive trophic level in the 

aquatic community.

This investigation was designed to determine the 96-h toxicity of analytical and field 

grade TFM to unialgal cultures and to determine the effects of TFM on growth and production of selected diatoms and green and 

blue-green algae. Growth was measured by 

making daily optical density readings and 

cell counts of nonfilamentous species, and 

determining maximum specific growth rates 

and total biomass produced by each culture.

METHODS

Unialgal cultures of four green algae, four 

blue-green algae, and two species of diatoms 

were obtained from the sources listed in 

Table 1. Several culture media were evaluated
4 Investigations in Fish Control 56: Fish and Wildlife Service

Table 1. Scientific names and sources of algal species

Species Source

Chlorophyta

Scenedesmus quadricauda (Turp.) Breb,

StigeocIonium tenue Kutz. 

Selenastrum capricornutum Printz 

Ghlorella pyrenoidosa Chick

Indiana Culture Collection, 

Indiana University

Indiana Culture Collection, 

Indiana University

National Eutrophication 

Research Program-EPA

Indiana Culture Collection, 

Indiana University

Cyanophyta

Gylindrospermum sp.

Anabaena flos-aquae (Lyngb.) 

De Brebisson

Nostoc linckia (Roth) Born, et Flash,

Anabaena cylindrica Lemmerman

Indiana Culture Collection, 

Indiana University

National Eutrophication 

Research Program-EPA

Michigan State University 

Culture Collection

Michigan State University 

Culture Collection

Chrys ophyta

Nitzschia sp.

Navicula pelliculosa (Breb.) Hilse

Indiana Culture Collection, 

Indiana University

Indiana Culture Collection, 

Indiana University

and the three that produced best growth 

of the three algal groups were used: Green 

algae were grown in ASM-1 medium (Gorham 

et al. 1964; also in Eberly 1967), blue-green 

algae in Alien and Arnon medium (Alien and 

Arnon 1955), and diatoms in the National 

Eutrophication Research Program medium

(Environmental Protection Agency [EPA] 

1971) with the addition of silica (10 mg/1). 

The chemical composition of each medium is 

shown in Table 2. The pH of each medium 

was adjusted to 7.5 t 0.2 by the addition of 

0.1N HC1 or 0.1N NaOH. Stock cultures were 

transferred to fresh media weekly to insure a
Maki, Geissel, and Johnson: Toxicity of TFM to 10 Species of Algae

Table 2. Chemical composition of synthetic algal nutrient media

Element

Major

N

P

Mg

S

C

Ca

Na

K

Cl

Si

Minor

B

Mn

Zn

Co

Cu

Mo

Fe

V

Concentration in culture (mg/2 -major; 

Mg/lt -minor)

NERPa 

with silica

4.200

0.186

2.904-

1.911

2.143

1.202

11.001

0.469

6.611

10.00

32.5

115.374

15.691

0.354

0.004

2.878

33.051

Alien and 

Arnon (1955)

0.270

7.790

3.081

4.353

1.155

2.559

12.735

20.735

23.585

62.5

62.4

6.3

1.2

2.5

15.0

483.0

1.30

ASM-1

4.753

6.201

7.080

4.742

2.384

8.073

76.970

7.812

96.465

431.0

333.0

210.0

3.71

0.372

67.0

188.0

National Eutrophication Research Program,
6 Investigations in Fish Control 56: Fish and Wildlife Service

continuous supply of cells in logarithmic 

growth phase to serve as inoculum for the 

toxicity tests.

Procedures and statistical analysis of the 

data followed the basic recommendations of 

the National Eutrophication Research Program (EPA 1971). All toxicity tests were 

carried out in a walk-in environmental chamber where incubation conditions were constant 

throughout the experiments. Cultures were 

maintained at 23 t 1C under continuous, 

cool-white fluorescent lighting of 400 (± 10%) 

foot-candles. The inoculum for each test was 

adjusted to yield an initial cell concentration 

of about 1.0 x 10 4 cells/ml. Tests were conducted in 250-ml polyurethane-stoppered 

Erlenmeyer flasks with 60 ml of autoclaved 

medium on reciprocating shakers operating 

at 80 oscillations per minute.

Preliminary tests to define the range of 

toxicity for several species established the 

50% effect at 5 to 10 mg/1; therefore, concentrations of 2.5, 5.0, 7.0, 8.0, 10.0, and 

15.0 mg/1 and a control were used for each 

toxicity test. Field grade and analytical grade 

TFM were tested simultaneously, with three 

replicates for each toxicant concentration; 

thus 42 culture flasks were used for each 

test. All data presented represent an average 

of two or three of these toxicity tests.

Purified TFM(A) (95% Aldrich lot #060217) 

and field grade (#1414, 35.7%, Drum 279 of 

Batch 6) TFM were used in all tests. Concentrations were based on active TFM rather 

than on the formulation. Primary and secondary stock solutions of the toxicant were made 

up in acetone and water, respectively, and 

periodically renewed throughout the experiments. All test flasks received 7.8 mg acetone per 60 ml culture (0.130 gm/1) from the 

stock solutions. Preliminary tests with three 

times this acetone concentration produced no 

measurable effects on algal growth.

Each toxicity test was conducted for 96 h to 

establish the lethal concentrations. The growth 

of the algae was determined spectrophoto~ 

metrically at 680 nm on a Beckman DB spectrophotometer or a Bausch and Lomb Spectronic 20. The unicellular forms were counted

directly under a microscope with a hemacytometer.

The specific growth rate as defined by the 

following formula was calculated for all concentrations of each toxicity test.

u = days _ i

t 2 -

where X2 = biomass concentration at end of 

selected time interval

Xj = biomass concentration at beginning 

of selected time interval

t 2 - tj = elapsed time in days between 

selected determinations of biomass (EPA 

1971).

We calculated linear regressions of the effects of both analytical and field grade TFM 

on maximum standing crop of algae, employing the following function:

dry weight, y = a + b (TFM concentration)

Biomass was expressed in milligrams dry 

weight of each culture.

Maximum standing crop was determined 

gravimetrically for each test flask at the end 

of the 96-h toxicity test by filtering a measured portion of algal suspension through a 

tared Millipore ® filter type A A with 

0.80-/um pores. Filters were oven-dried at 

80 C for 24 h, cooled in a desiccator, and 

weighed. A correction factor was developed 

and subtracted to correct for loss of weight 

of the filters during washing. Toxicant concentration was checked at the beginning and 

end of each toxicity test by comparison with a 

standard curve on a Klett-Summerson colorimeter. Concentrations of TFM in the test 

solutions and pH at the termination of the test 

were checked on a portion of the medium 

filtered free of algal cells.

Slight decreases in TFM concentration were 

observed at the termination of each test, presumably due to absorption and uptake of TFM
Maki, Geissel, and Johnson: Toxicity of TFM to 10 Species of Algae 7

by algal cells. Changes in pH between initiation and termination of the test were of the 

order of 0.1-0.4 pH units and were not consistently different between media.

The optical density data were analyzed by 

the methods of Litchfield and Wilcoxon (1949) 

for the evaluation of median effective concentrations (EC50) and establishment of 95% 

confidence limits. The EC50 is the TFM concentration that causes a 50% inhibition of 

a3gal growth when compared with control cultures growing simultaneously in the absence 

0* the lampricide.

RESULTS

Toxicity Tests

The inhibition of growth by TFM differed for 

the different algal taxa (Table 3; Fig. 1). In 

general, it was lowest for the blue-green 

algae, intermediate for the green algae, and 

most severe for diatoms (Fig. 2). Exceptions 

were the unicellular green alga Chlorella 

pyrenoidosa, which appeared to be very resistant to TFM (EC50 greater than 15 mg/1 

of TFM) and the filamentous blue-green algae 

Anabaena flos-aquae and Anabaena cylindrica, 

which were highly susceptible to the lampricide (ECSO's, 1.8 to 4.7 mg/1 of TFM). Although the EC50 values were slightly lower 

for field grade than for analytical TFM for 

each species tested, the differences were 

statistically significant only for the highly 

sensitive diatom Nitzschia sp. However, the 

data suggest that the additive N, N-dimethylformamide in field grade material may augment the toxicity of the compound.

Specific Growth Rates

The maximum specific growth rate (jumax 

see Table 4) for individual species and concentrations at any time during incubation occurred on the second or third day of each 

toxicity test.

The values reflect the toxicity data presented in Table 3; the growth rates were generally best in control cultures and gradually

declined as TFM concentration increased. 

Growth rates were more severely depressed 

in cultures with field grade than with analytical grade TFM. Negative growth rates for 

the diatoms Nitzschia sp. and Navicula 

pelliculosa indicate mortality of the inoculum 

at the higher dose levels on the second day of 

exposure.

In several of the cultures in 2.5 mg/1 of 

analytical grade TFM, growth was apparently 

stimulated initially, since the^imax exceeded 

that of the controls. However, the cell density 

and standing crop at the end of the 4-day 

toxicity test were lower than in the controls, 

indicating that growth was suppressed at 

these low concentrations.

Regression analysis of toxicant concentration 

and maximum standing crop

The maximum standing crop for each test is 

defined as the weight of the total algal biomass 

at the end of the 4-day toxicity test, after filtration through a tared Millipore filter and 

oven-drying at 80 C for 24 h. Linear regressions of the effects of analytical and field 

grade TFM on maximum standing crop of 

algae were produced for the species whose 

growth was not severely limited by relatively 

low TFM concentrations.

The equations serve to establish a degree 

of linearity of response to the proportional 

increases in toxicant concentration (EPA 

1971).

The linear relation between TFM concentration and maximum standing crop describes 

the effect of increasing TFM concentration, as 

evidenced by the relatively high regression 

coefficients for most species (Table 5). However, for species whose growth is severely 

limited by relatively low concentrations of the 

lampricide, a curvilinear relation may best 

describe the data. The gravimetric determination of growth (Table 5) is not as sensitive 

a measure of growth as is the optical density 

reading (Fig. 1) for even though the optical 

density data indicate measurable growth, the 

gravimetric data do not.
8 Investigations in Fish Control 56: Fish and Wildlife Service

Table 3. Growth inhibition of TFM9" to algae as measured by optical density

Species

96-h EC50 and 95$ confidence 

interval (mg/e)

Field grade Analytical grade

Chlorophyta 

Scenedesmus quadricauda

Stigeoclonium tenue

Selenastrum capricornuturn 

Chlorella pyrenoidosa 

Cyanophyta

4.2 

2.21-7.98

4.7 

3.24-6.81

5.5 

4.14-7.32

4.5 

2.81-7.20

6.2 

4.43-8.68

8.6 

5.93-12.5

Cy 1 indr ospermum sp. 

Anabaena flos-aquae

Nostoc linckia

Anabaena cylindrica

Chrysophyta 

Nitzschia sp.

Navicula pelliculosa

9.0 

7.50-10.8

3.6 

2.40-5.40

9.2 

8.00-10.6

1.8 

0.82-3.96

1.2 

0.71-2.04

3.2 

2.00-5.12

9.9 

3.96-24.8

4.2 

2.47-7.14

. 9.8 

6.12-15.7

4.7 

2.90-7.61

3.6 

2.57-5.04

5.2 

3.71-7.28

Concentration based on active TPM.

The higher toxicity of the field formulations 

is again shown for all three groups of algae by 

the lower y-intercept and depressed regression of the total biomass produced in the field 

grade toxicity tests. The most sensitive species are distinguished by their high intercept 

values and relatively shallow slopes.

Cell Counts

A hemacytometer was employed for direct 

microscopic counting of Scenedesmus 

quadricauda, Selenastrum capricornutum, and 

Chlorella pyrenoidosa. Accurate counts of the 

other species were not possible. Filamentous
OPTICAL DENSITY 

g S

OPTICAL DENSITY 

§b o -

01 ___________W O
10 Investigations in Fish Control 56 : Fish and Wildlife Service

hlorello pvrenoidoso 

Analytical Grade

1. Continued

species wfitte diHjcult to count, and the diatoms 

tended to g^vr fn small, tightly packed 

clumps. Attempts to equally disseminate the 

filamentous forms and clumping species by 

blending or dispelling them through a syringe 

were unsuccessful. The counts for the three 

species yielded an excellent representation of 

the inhibition of growth by TFM (Fig. 3).

Mortality Tests

Inasmuch as the toxicity tests showed a 

definite suppression of algal growth that was 

directly correlated with TFM concentration, 

we designed a series of experiments to determine whether the growth suppression was a 

result of (1) toxicant-induced mortality of 

algal cells, (2) a temporary growth suppression of the cells at the end of the 4-day toxicity test, or (3) a selective elimination of a 

proportion of the new cells being produced 

which gave the indication that no new growth 

was occurring. We established replicate toxicity tests using the standard technique and 

the TFM concentrations of 15 and 30 mg/1, 

which exceeded the 96-h lethal concentrations

Anabaena flos-aquae

Stiaeoclonium tenue

0 2.5 5.0 7.0 8.0 9.0 10.0 

TFM CONCENTRATION (mg/1)

Fig. 2. Relative growth of representative algal 

species grown in increasing concentrations of 

analytical grade TFM. expressed as a percentage of control optical density at 96 h.

for all species except Chlorella pyrenoidosa. 

After the cells had been exposed at these 

concentrations for 96 h, they were filtered 

free of the dosed medium and resuspended in 

TFM-free medium. This procedure simulated 

a natural situation in which an algal population is exposed to high lampricide concentrations for a relatively long period (4 days) in a 

bay or backwater at the mouth of a stream 

being treated for lamprey control. No growth 

was observed in any of the cultures during 

the initial 96-h exposure to 30 mg/1, and 

Chlorella pyrenoidosa was the only species to 

produce measurable growth at 15 mg/1 of 

TFM.
Maki, Geissel, and Johnson: Toxicity of TFM to 10 Species of Algae 11

Table 4. Maximum specific growth rates (/max average of three replicates) 

of 10 algal species at different concentrations of TFM

Species, and 

grade of TFM

TFM Concentration (mg/C of active ingredient)

2.5 5.0 7.0 10.0 15.0

Scenedesmus quadricauda

Analytical

Field 

Stigeoclonium tenue

Analytical

Field 

Selenastrum capricornutum

Analytical

Field 

Chorella pyrenoidosa

Analytical

Field 

Cylindrospermum sp.

Analytical

Field 

Anabaena flos-aquae

Analytical

Field 

Nostoc linckia

Analytical

Field

Anabaena cylindrica

Analytical

Field 

Nitzschia sp.

Analytical

Field 

Navicula pelliculosa

Analytical

Field

1.230 0.867 0.815 0.351 0.231 0.239 0.201

1.001 1.179 0.405 0.351 0.296 0.317 0.131

0.621 0.892 0.900 0.215 0.131 0.049 0.077

0.718 0.476 0.270 0.166 0.140 0.049 0.000

1.751 1.825 1.959 1.839 1.546 1.405 1.497

1.649 1.533 1.358 0.990 0.875 0.604 0.336

1.619 1.445 1.351 1.440 1.834 1.488 1.660

1.413 1.449 1.493 1-470 1.479 1.475 1.460

1.685 1.335 1.683 1.332 1.197 1.435 1.327

1.224 1.264 1.163 0.486 0.836 0.767 0.692

0.784 0.900 0.667 0.582 0.412 0.095 0.093

0.673 0.683 0.270 0.068 0.034 0.033 0.025

0.811 0.747 0.723 0.658 0.593 0.631 0.525

0.713 0.846 0.775 0.673 0.599 0.536 0.451

1.212 1.241 0.728 0.315 0.515 0.195 0.221

1.037 0.300 0.255 0.298 0.182 0.374 0.270

0.788 1.808 0.668 -0.930 -1.098 -

1.037 0.104 0.104 -1.040 -1.099 -1.945 1-252

1.229 1.253 1.184 0.693 0.095 -0.241 -0.105

1.239 1.209 0.489 0.322 -0.134 0.255 0.104
Table 5. Maximum standing crop (dry weight in milligrams) of 10 species of algae after 

different TEM concentrations. Regression equations are given for species showing a 

Species, and 

grade of TJM

Scenedesmus quadricauda

Analytical

Field

Stigeoclonium tenue

Analytical

TEM Concentration (ing/ fi of active ingredient)

 

0 2.5 5.0 7.0 8.0 10.0 15.0

8.8 7.0 4.4 0.4 0.6 0.4 0.0 

4.6 6.8 2.0 0.0 2.0 0.0 0.0

%

9.6 7.6 10.0 1.4 1.4 1.0 1.0 

Field

Selenastrum capricornutum

Analytical 4.0

Field

9.4 9.2 1.6 1.2 1.0 1.0 1.2 

4.2 3.8 2.8 1.6 1.2 0.0 

3.8 3.0 1.8 0.2 0.0 1.2 0.0 
Table 5. Maximum standing crop (dry weight in milligrams) of 10 species of algae 

to different TFM concentrations. Regression equations are given for speci

line ar re spons e Continued.

Species, and 

grade of TEM

Chlorella pyrenoidosa

Analytical

Field 

Cyl indrospermum sp.

Analytical

Field

Anabaena flos-aquae

Analytical

Field

Nostoc linckia

Analytical 

Field

TFM Concentration

0 2.5 5.0

5.6 5.2 5.4

5.2 4.4 5.4

12.0 8.0 9.2

5.4 6.8 5.0

10.3 8.8 6.8

11.2. 8.2 3-0

8.8 6.2 5.6 

5.6 6.4 5.6

(mg/2 of active

7.0 8.0

5.2 5.4

5.0 5.2

6.2 5.4

3.6 4.0

2.2 1.4

2.6 2.4

5.0 4.6 

2.6 2.4

ingredient)

10.0 15.0

5.0 5.0

4.8 4.6

2.4 5.2

1.6 0.8

1.4 1.4

2.2 2.0

2.6 2.8 

0.4 0.0
Table 5. Maximum standing crop (dry weight in milligrams) of 10 species of algae a

to different TFM concentrations. Regression equations are given for specie

linear response Continued.

Field

Navicula pelliculosa 

Analytical

Field

4.2 0.2 0.2 0.2 0.0 0.0 0.0

8.4 6.6 5.0 1.0 0.0 0.0 0.0 

6.8 3.8 0.0 0.0 0.0 && 0.0

Species, and 

grade of TFM

Anabaena cylindrica

Analytical

Field 

Nitzschia sp.

Analytical

TFM Concentration (mg/B of active ingredient) 

 : 

0 2.5 5.0 7.0 8.0 10.0 15.0 

3.0 1.8 0.4 0.0 0.0 0.0 0.0 

3.8 0.0 0.0 0.0 0.0 0.0 0.0 

4.0 3.6 1.4 0.4 0.2 0.0 0.0 
Maki, Geissel, and Johnson: Toxicity of TFM to 10 Species of Algae 15

Selenostrum copricornutum Selenostrum copricornutum

Scenedesmus quodricoudo 

Field Grade

- Chlorella pyrenoidoso 

Analytical Grade

10'

10=

Chlorella pyrenoidasa 

Field Grade

I 2 

DAYS

I 2 

DAYS

Fig. 3. Cell numbers in algal cultures grown in 

selected concentrations of analytical and field 

grade TFM.

which did not grow after the 96-h exposure to 

30 mg/1 of TFM.

The remaining possibility that the cells 

were indeed growing in the high concentrations, but that the toxicant was selectively 

eliminating a proportion of the new cells being produced remains unresolved. However, 

microscopic examinations of TFM-exposed 

algal cells while they were being counted 

during the toxicity tests tended to indicate that 

no vegetative reproduction was occurring, and 

that TFM was producing severe sublethal effects on the integrity of the chloroplast and 

other cell inclusions. Misshaped and shrunken 

chloroplasts were commonly observed in 

cells exposed to the higher concentrations of 

TFM, but normal growth and reproduction 

ensued soon after the cells were resuspended 

in toxicant-free medium.

Another possibility that warranted further 

investigation was that the color imparted to 

the algal growth medium by release of the 

phenolate ion from TFM selectively shades 

out a portion of the light spectrum and thus 

inhibits growth of the cultures. To test this 

hypothesis, we added sufficient food coloring 

to the growth medium to produce a yellowgreen color that absorbed at 475 Klett Units 

an absorption similar to that of a 30 mg/1 

solution of analytical grade TFM. Both species tested Anabaena cylindrica and 

Selenastrum capricomutum produced 

greater cell densities in 96 h in the flask to 

which food coloring had been added than in 

the control flasks. Although the absorption 

spectra of TFM and food coloring may not be 

exactly equivalent, we assumed on the basis 

of this test that shading from the colors of 

the phenolate ion of TFM did not limit growth.

After resuspension in toxicant-free medium, 

each alga showed little or no growth for 2 to 3 

days. Soon after this lag, each culture grew, 

and cell density reached that in the controls 

after an average of 12 days, indicating that 

viability had not been destroyed at these high 

concentrations but that the ability to grow and 

reproduce was severely limited by the presence of the toxicant. The exception in this test 

was the sensitive diatom Navicula pelliculosa,

DISCUSSION

Tests of the toxicity of analytical (95%) and 

field grade (35.7%) TFM to unialgal cultures 

of representative green and blue-green algae 

and diatoms showed that field grade material 

tended to exert the greater toxicity, when expressed on the basis of percentage active ingredient; 96-h 50% effect values generally 

were 2 to 3 mg/1 lower for field grade than
16 Investigations in Fish Control 56; Fish and Wildlife Service

for analytical grade material. Although the 

values were not statistically different, the 

tendency agrees with tendencies demonstrated 

in insects and fish.

The 96-h 50% effect levels ranged from 1.2 

to more than 15 mg/1 for field grade TFM 

and from 3.6 to more than 15 mg/1 for analytical grade material. The most pronounced 

effects of the toxicant were observed in the 

diatom cultures, where 50% tolerance limits 

ranged from 1.2 to 5.2 mg/1. Intermediary 

effects were observed among the green algae, 

where toxicity values generally ranged from 

4 to 8 mg/1; the exception was Chlorella 

pyrenoidosa which was resistant to concentrations greater than 15 mg/1. Morgan (1972) 

found that a unicellular green alga, 

Chlamydomonas reinhardtii, grew well at 20 

mg/1 of Aroclor 1242, a concentration normally lethal to other aquatic life. Zweig et al. 

(1968) found Chlorella pyrenoidosa to be 

relatively resistant to low concentrations of 

the herbicides diquat and 1,4-benzoquinone; 

the effects of the herbicides were termed 

algistatic rather than algicidal.

We found the more TFM-resistant species 

among the blue-green algae although two 

species of Anabaena had ECSO's which approached those of the sensitive diatom species. This high susceptibility of Anabaena is 

unexplained.

Lampricide sensitivity differed little between species; i.e., in all of them, progressively lower growth rates were correlated 

with progressively higher toxicant levels. 

Several of the species expressed a higher 

Atmax at 2.5 mg/1 of TFM than did the controls, although the total biomass yield at the 

end of the 96-h test did not exceed the yield of 

control cultures. Stadnyk et al. (1971) reported similar increased growth rates and 

cell numbers, but not biomass, for cultures of 

Scenedesmus quadricauda exposed to 0.1 and 

1.0 mg/1 of organochlorine insecticides. The 

TFM may have been metabolized at low concentrations, either by the algal cells themselves or by contaminating bacteria. Although 

Klett Unit absorbance of filtered media did not 

change after any of the tests, the colorimeter

may have been insufficiently sensitive to detect metabolism of the lampricide.

The present study indicates that residues of 

TFM inhibited algal growth at concentrations 

that might be developed during stream treatments. The inhibition was only temporary, 

however, and even exposures to the high concentration of 30 mg/1 for 4 days did not decrease the viability of algal cells when they 

were resuspended in toxicant-free water. 

Although metabolic activity such as oxygen 

evolution or carbon assimilation were not 

measured, an effect on these activities is 

implied in the total reproductive capacity of 

the algal cultures. Further experimentation 

may yield an understanding of the nature of 

the temporary growth inhibition.

CONCLUSIONS

1. The lampricide TFM inhibited growth of 

all algal species tested. Concentrations 

of less than 10 mg/1 caused a 50% inhibition of growth in all species except 

Chlorella pyrenoidosa, which was resistant to test concentrations up to 15 

mg/1.

2. Diatoms were the most sensitive species 

tested; 50% inhibition of growth occurred 

at concentrations of 1-4 mg/1 of TFM. 

The green algae were intermediate in 

sensitivity. In blue-green algae, the 

least susceptible group, 50% inhibition of 

growth generally occurred near 10 mg/1 

of TFM.

3. Field grade TFM generally tended to be 

more toxic than analytical grade material.

4. TFM does not appear to be algicidal within the range of concentrations tested but 

rather produces a temporary algistatic 

inhibition of growth.

ACKNOWLEDGME NTS

This investigation was conducted under 

Contract 14-16-0008-651, U.S. Department of 

Interior, Fish and Wildlife Service, Bureau of
Maki, Geissel, and Johnson: Toxicity of TFM to 10 Species of Algae 17

Sport Fisheries and Wildlife. The assistance 

of Joseph Hunn, Contract Officer, and the cooperation of staff members of the Michigan 

State University Pesticide Research Center is 

gratefully acknowledged.

Michigan Agricultural Experiment Station 

Journal Article Number 6580.

REFERENCES

Alien, M. B., and D. I. Arnon. 1955. Studies on 

nitrogen-fixing blue-green algae. I. Growth and 

nitrogen fixation by Anabaena cylindrica Lenn. 

Plant Physiol. 30:366-372.

Eberly. W. R. 1967. Problems in the laboratory culture of planktonic blue-green algae. Pages 7-34 in 

Environmental requirements of blue-green algae; 

proceedings of a symposium, Sept. 23-24. 1966. 

U.S. Department of Interior. Federal Water Pollution Control Administration. October 1967. Corvallis. 

Oregon.

Environmental Protection Agency. 1971. Algal 

assay procedure bottle test. National Eutrophication Research Program. Corvallis, Oregon.

Gorham, P. R., J. McLachlan, V. T. Hammer, and 

W. K. Kirn. 1964. Isolation and culture of toxic 

strains of Anabaena flos-aquae (Lyngb. de Breb.). 

Int. Vert. Theor. Angew. Limnol. Verh. 15:796-804.

Haas, R. C. 1970. The effect of lamprey larvicide on 

the bottom fauna and periphyton of the Chocolay 

River, Marquette County, Michigan. Mich. Dep. Nat. 

Resour., Res. Dev. Rep. 200. 66 pp.

Howell. J. H. 1966. The life cycle of the sea lamprey 

and a toxicological approach to its control. Pages 

263-270 in R. T. Smith, P. A. Miescher, and 

P. M. Good, eds. Phylogeny of immunity. University 

of Florida Press, Gainesville, Florida.

Josephs, M. J. 1961. Control of aquatic plants. U.S. 

patent 3,004,843. Filed Aug. 10, 1959. Patented 

Oct. 17, 1961. 3 pp.

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

method of evaluating dose-effect experiments. 

J. Pharmacol. Exp. TTier. 96:99-113.

Morgan, J. R. 1972. Effects of Arocolor 1242® <a 

polychlorinated biphenyl) and DOT on cultures of 

an alga, protozoan, daphnid, ostracod, and guppy. 

Bull. Environ. Contam. Toxicol. 8:129-137.

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

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

larvicide. U.S. Bur. Sport Fish. Wildl. Invest. 

Fish Control No. 44. 31 pp.

Stadnyk, L.. R. S. Campbell, and B. T. Johnson. 1971. 

Pesticide effect on growth and 14C assimilation in 

a freshwater alga. Bull. Environ. Contam. Toxicol. 

6:1-8.

Zweig, G.§ J. E. Hitt, and R. McMahon. 1968. Effect 

of certain quinones. diquat, and diuron on Chlorella 

pyrenoidosa_ Chick. (Emerson strain). Weed Sci. 

16:69-73.

INVESTIGATIONS IN FISH CONTROL

57. Acute Toxicities of 3-TrifluoromethyS-4-nitrophenol 

(TFM) and 2',5-Dichloro-4'-nitrosalicylanilide 

(Bayer 73) to Larvae of the Midge Chironomus tentans

By Joseph A. Kawatski, Margaret M. Ledvina, 

and Carl R. Hansen, Jr.

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. 1975
CONTENTS

Page 

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

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

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

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

Discussion ..................................... 4

Conclusions .................................... 6

Acknowledgment ................................. 6

References. .................................... 6
ACUTE TOXICITIES OF 3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM)

AND 2',5-DICHLORO-4'-NITROSALICYLANILIDE (BAYER 73) TO LARVAE

OF THE MIDGE (CHIRONOMUS TENTANS)

By Joseph A. Kawatski, Margaret M. Ledvina, and Carl R. Hansen, Jr.

Department of Biology, Viterbo College

La Crosse, Wisconsin

ABSTRACT

The toxicants 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro4'-nitrosalicylanilide (Bayer 73) were tested individually and together for 

toxicity to fourth instar Chironomus tentans in laboratory static tests at 

22 t 1 C. The 24-h EC50 (immobility) values in soft water (total hardness, 

40-48 mg/jg as CaCO3 ) were 1.55 mg/K of 95.7% TFM, 0.570 mg/fi of 

70% Bayer 73, and 0.658 mg/fi of total toxicant for a 98:2 mixture of TFM 

and Bayer 73. As water hardness increased, toxicity of the materials 

individually and in mixtures decreased. Generally, the toxic effect of 

mixtures of TFM and Bayer 73 was additive; synergistic toxicity was 

apparent only in soft water at exposures of 24 to 72 h. Immobility was not 

coincident with death in chironomids. Considerably greater concentrations of toxicant were required to kill than to immobilize.

INTRODUCTION

The toxicant 3-trifluoromethyl-4-nitrophenol 

(TFM) has been used extensively and successfully to control the sea lamprey (Petromyzon 

marinus) in the Great Lakes. Authorization for 

the continued use of the chemical will depend 

on the outcome of current research designed 

to clarify the effects of TFM on a variety of 

nontarget organisms (Schnick 1972). The molluscicide Bayer 73 (2-aminoethanol salt of 

2',5-dichloro-4-nitrosalicylanilide) is applied 

with TFM to produce a 2% mixture, i.e., 98 

parts of TFM (95.7%): 2 parts Bayer 73 (70% 

W.P.), here termed TFM: 2B. This mixture 

has an apparent synergistic effect on lamprey 

larvae (Howell et al. 1964). Inasmuch as invertebrates constitute a vital portion of 

aquatic food webs, the elucidation of the toxicities of TFM and Bayer 73 to invertebrates is 

essential.

Among the invertebrates in most aquatic 

food chains, insect larvae, including midges, 

represent one of the most important links at a 

relatively low trophic level. We report here on 

the acute toxicities of TFM, Bayer 73, and

TFM-2B to laboratory-reared, fourth instar 

populations of Chironomus tentans Fabricius, 

a widely distributed benthic midge and an 

important fish food organism. For reference, 

we also determined the toxicity of antimycin A 

against this chironomid.

MATERIALS AND METHODS

The U.S. Fish and Wildlife Service, Fish 

Control Laboratory, La Crosse, Wisconsin, 

provided the following materials for this 

study: TFM (95.7% 3-trifluoromethyl-4- 

nitrophenol, Aldrich Chemical Co., Lot No. 

060217); Bayer 73 (70% 2-aminoethanol salt of 

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

powder, Chemagro Corp., Lot No. 8059410); 

antimycin A (95.5%, Ay erst Laboratories, Lot 

No. 1294-L); and reconstituted water. Soft, 

hard, and very hard waters, with respective 

total hardnesses of 40-48, 160-180, and 280- 

320 (as mg/£ CaCO3 ), were used in the toxicity tests. These waters were further characterized by Marking (1970). The food and 

substrate mixture consisted of ground Trainers Dog Rewards® (Horlick's Corp., Racine,
Investigations in Fish Control 57: Fish and Wildlife Service

Wisconsin) and macerated paper hand towels 

(Crown Zellerback Corp., San Francisco, 

California). This mixture contained no detectable TFM or Bayer 73 residues as determined 

by gas-liquid chromatographic analysis.

Stock cultures of C. tentans were propagated 

in the laboratory in 5-, 20-, and 400-liter 

glass or fiberglass aquaria, in soft, reconstituted water at a temperature of 22 + 1.5 C, 

under a 16-h photoperiod of mixed fluorescent 

and incandescent light. Food and substrate 

were added periodically to sustain growth and 

reproduction. Continuous gentle aeration was 

provided. This rearing system was essentially 

that of Derr and Zabik (1972).

About 48 h before each toxicity test, we 

transferred 10-20 organisms individually, with 

a curved blunt probe, into separate beakers 

containing 900 m£ of well-aerated test water. 

The toxicants, dissolved in 50% acetone (in 

water), were then introduced into the exposure 

vessels to produce the appropriate test concentrations. The total amount of acetone added 

to each vessel and to control systems was 

0.5 m2. The criterion for evaluating toxicant 

effect was immobility, noted after 8, 24, 48, 

72, and 96 h of exposure. A test animal was 

considered immobile when it failed to move 

its body, jaws, or anal gills after it was 

touched lightly with a glass probe. The water 

was not aerated during exposures. Toxicity 

tests with TFM, Bayer 73, and TFM-2B were 

replicated at least three times for each of 

three water hardnesses at 22 ± 1 C. Using the 

Litchfield and Wilcoxon (1949) method, we 

calculated the ECSO's from the combined data 

of at least three independent tests. (The EC50 

is defined as the concentration of toxicant, 

based on total formulation weight, that immobilized 50% of the test organisms within 

the prescribed period of exposure.) Results of 

tests in which more than 20% of the control 

organisms died were discarded. From 114 to 

590 animals were used to determine the 

ECSO's for each chemical or combination of 

chemicals. The ECSO's derived for TFM-2B 

were based on total toxicant, i.e., the sum of 

TFM and Bayer 73.

The toxicity of TFM-2B was compared with 

the toxicities of the two lampricides alone by

calculation of additive indices as described by 

L. L. Marking and V. K. Dawson (in preparation). The significance of index values is 

determined from ranges for the indices, using 

95% confidence limits for the EC50 values. If 

the range for an index is above zero, the 

toxicity of the combined chemicals is synergistic; if the range is below zero, the toxicity 

is antagonistic.

RESULTS

Water quality had a direct effect on the toxicity of TFM, Bayer 73, and TFM-2B against 

C^. tentans (Table 1.) Hard water with high 

pH (8.0-8.4) decreased the toxicity of the 

chemicals, whereas soft water with a lower 

pH (7.2-7.6) increased it. The influence of 

water quality was less pronounced with Bayer 

73 than with TFM. In comparison with TFM, 

the toxicity of Bayer 73 to Q tentans was 2 to 

3 times greater in soft water and 6 to 12 times 

greater in hard water.

At most water hardnesses and exposure 

periods, the addition of 2% of Bayer 73 reduced 

the amount of TFM required to immobilize 

chironomids. However, the ranges of additive 

indices for TFM-2B indicated that synergistic 

toxicity occurred only in soft water and then 

only at three intermediate exposure periods 

(Table 2). Additive index ranges overlapped 

zero in all water hardnesses at 8- and 96-h 

exposures; when test water was hard or very 

hard, the toxic effect of TFM-2B was additive.

Antimycin A was used as a reference chemical to determine the sensitivity of our laboratory population of jC. tentans. In soft water, 

the 96-h LC50 and 95% confidence limits were 

determined to be 0.146 (0.095-0.224) Mg/£.

DISCUSSION

Howell et al. (1964), who determined the 

lethal concentrations of TFM to lamprey 

larvae in water ranging from soft with a pH 

of 7.3 to very hard with a pH 8.5, reported 

that toxicity decreased as hardness and pH 

increased. They found, however, that even in 

hard water the amount of TFM-2B required to
Kawatski et al.: Acute Toxicities of TFM and Bayer 73 to Larvae of the Midge 5

Table 1. Toxicities of TFM, Bayer 13, and a 98:2 mixture of TFM and Bayer 73 to fourth instar Chironomus

Chemical and 

water hardness 

(mg/g as CaC03 )

TFM

Soft 

(40-48)

Hard 

(160-180)

Very hard 

(280-320)

Bayer 73

Soft 

(40-48)

Hard 

(160-180)

Very hard 

(280-320)

Mixture, TFM, and 

Bayer 73 (98:2)

Soft 

(40-48)

Hard 

(160-180)

Very hard 

(280-320)

a EC50 (immobility)

Table 2. Additive

Water hardness 

(as mg/c CaCo3 )

Soft 

(40-48)

Hard 

(160-180)

Very hard 

(280-320)

tentans in waters o:

8 h

1.65 

1.45-1.88

6.47 

5.66-7.39

14.3 

13.1-15.6

0.744 

0.550-0.998

1.01 

0.712-1.43

2.01 

1.45-2.79

1.38 

1.18-1.61

4.33 

3.76-4.99

11.8 

10.7-13.1

values for mixture are

C different ranges of hardness at 22 + 1 C

EC50 (mg/g) a and 95$ confidence intervals at:

24 h 48 h 72 h

1.55 0.968 0.790 

1.32-1.83 0.815-1.15 0.631-0.989

4.54 2.53 1.19 

4.06-5.08 2.13-3.01 0.937-1.51

10.0 6.60 3.49 

8.09-12.4 5.47-7.97 2.84-4.29

0.570 0.367 0.347 

0.458-0.710 0.289-0.467 0.257-0.469

0.640 0.457 0.360 

0.501-0.817 0.352-0.593 0.275-0.471

0.814 0.536 0.374 

0.630-1.05 0.417-0.690 0.286-0.490

0.658 0.400 0.358 

0.474-0.913 0.312-0.513 0.283-0.453

2.59 1.52 0.910 

1.76-3.81 1.19-1.95 0.749-1.11

7.03 4.94 3.50 

5.89-8.39 4.14-5.89 2.81-4.36

expressed as total toxicant (TFM plus Bayer 73) .

indices for the toxicity of a 98:2 mixture of TFM and Bayer 73 against 

tentans in selected harndesses at 22 + 1 C

8 h

+0.167 

-0.147-f 0.566

+0.348 

-0.004-+0.814

+0.080 

-0.161-f 0.335

Additive indices and their ranges at:

24 h 48 h 72 h

+1.28 +1.34 +1.15 

+0.930-+2.74 +0.533-+2.58 +0.354-+2.42

+0.562 +0.526 +0.250 

-0.072-4-1.61 -0.008-fl.34 -0. 242 -f 0.931

+0.161 +0.900 -0.170 

-0.283-+0.731 -0.338-+0.590 -0.809-+0.322

96 h

0.534 

0.415-0.688

0.998 

0.854-1.17

2.10 

1.65-2.67

0.228 

0.171-0.304

0.295 

0.221-0.393

0.353 

0.261-0.477

0.328 

0.262-0.416

0.657 

0.516-0.821

2.79 

2.36-3.29

Chironomus

96 h

+0.585 

-0.031-+1.56

+0.450 

-0.016-+1.18

-0.460 

-1.21-f 0.036

Indices with ranges less than zero suggest antagonism; indices with ranges that overlap zero suggest 

additive toxicity; and indices with ranges, greater than zero suggest synergism.
Investigations in Fish Control 57: Fish and Wildlife Service

kill lampreys was less than the amount of 

TFM alone required to kill them. B. R. Smith 

(1967) also observed that the toxicity decreased as water hardness increased when 

Bayer 73 and TFM-2B were tested against 

lampreys and rainbow trout (Salmo gairdneri), 

and that the degree of synergism decreased 

as water hardness and pH increased. Our data 

and those of Kawatski (1973) show that the 

response of midges and ostracods is similar 

to that of fish.

Some chironomids that were immobilized 

by TFM and appeared dead revived when they 

were placed in toxicant-free water. Postexposure observation enabled us to establish 

that, for TFM in soft water, the 8-h LC50 was 

12 times greater than the immobility-based 

8-h EC50. The disparity between EC50 and 

LC50 values resides with the difficulty in ascertaining whether a test animal is immobile, 

moribund, or dead. However, as exposure 

time lengthens, dead chironomids are more 

easily recognized, and the difference between 

the ECSO's and LCSO's in longer exposures 

(24-96 h) is much smaller.

Recovery of test organisms, especially 

after short-term exposure, was not surprising 

because earlier experiments (unpublished) in 

our laboratory showed that chironomids exposed to 14C-TFM rapidly biotransformed and 

excreted accumulated residue; this detoxication continued even after the organisms were 

immboilized. Because of these observations 

and the difficulty in defining death, particularly in small and/or normally sluggish invertebrates, it is clear to us that in shortterm exposures LCSO's can be accurately 

determined only if the affected organisms are 

held in toxicant-free water after the exposure 

period.

CONCLUSIONS

1. The toxicity of TFM, Bayer 73, and TFM2B against Chironomus tentans decreases 

as water hardness and pH increase.

2. Generally, the toxic effect of TFM and 

Bayer 73 in combination (TFM-2B) is additive; synergistic toxicity occurs only in

soft water at exposure times of 24 to 72 

hours.

3. Toxicant-induced immobility was not synonymous with death, as shown by the large 

percentage of toxicant-immobilized larvae 

that became active when they were placed 

in TFM-free water after exposure.

ACKNOWLEDGMENT

We thank S. K. Derr, Michigan State University, for supplying the stock culture of 

Chironomus tentans and the U.S. Fish and 

Wildlife Service, Fish Control Laboratory, 

La Crosse, Wisconsin, for supplying the toxicants and test water.

This study was funded by Contract 14-16- 

0008-644, Fish and Wildlife Service, U.S. 

Department of the Interior.

REFERENCES

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compounds in the aquatic environment: the uptake and 

distribution of [l,l-dichloro-2,2-bis(p-chlorophenyl) 

ethylene], DDE by Chironomus tentans Fabricius 

(Diptera: Chironomidae). Trans. Am. Fish. Soc. 

101(2):323-329.

Erkkila, L. F. 1962. Lamprey control and research in 

the United States. Pages 25-39 in Great Lakes Fishery Commission annual report for 1962.

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 lamprey larvicide. Great Lakes Fish. 

Comm., Tech. Rep. 8. 21 pp.

Kawatski, J. A. 1973. Acute toxicities of antimycin A, 

Bayer 73, and TFM to the ostracod Cypretta kawatai. 

Trans. Am. Fish. Soc. 102(4):829-83l.

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

J. Pharmacol. Exp. Ther. 96:99-113.

Marking, L. L. 1970. Juglone (5-hydroxy-l,4-napthoquinone) as a fish toxicant. Trans. Am. Fish. Soc. 99(3): 

510-514.

Marking, L. L., and V. K. Dawson. A method to assess 

the toxic or other effects of mixtures of chemicals. 

U.S. Fish Wildl. Serv., Fish Control Lab., La 

Crosse, Wis. 17 pp. (Unpublished)
Kawatski et al.: Acute Toxicities of TFM and Bayer 73 to Larvae of the Midge 7

Rye, R. P. 1972. The toxicity of two lamprey larvicides Smith, A. J. 1967, The effect of the lamprey larvicide

to selected aquatic Invertebrates. U.S. Fish Wildl. 3-trifluormethyl-4-nitrophenol, on selected aquatic

Serv0 Hammond Bay Biol. Sta., Millersburg, Mich. invertebrates, Trans. Am, Fish, Soc. 96(4):410-

14 pp. (Unpublished) 413.

Schnick, R. A. 1972. A review of literature on TFM Smith, B. R. 1967. Lamprey control and research in 

(3~trifluormethyl-4-nitrophenol) as a lamprey larvi- United States. Pages 28-45, in Great Lakes Fishery 

cide. U.S. Fish Wild. ServM Invest, Fish Control 44, Commission annual report for 1965. 

31pp.

INVESTIGATIONS IN FISH CONTROL

58. Acute Toxicity of the Lampricide

3-Trifluoromethyl-4-nitrophenol (TFM)

to Nymphs of Mayflies (Hexagenia sp.)

By Calvin R. Fremling

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. 1975
CONTENTS

Page 

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

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

Methods ............. .......................... 3

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

Discussion ...................................... 6

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

References ...................................... 7
ACUTE TOXICITY OF THE LAMPRICIDE 

3-TRIFLUOROMETHYL-4-NITROPHENOL(TFM)

TO NYMPHS OF MAYFLIES (HEXAGENIA SP.)

By Calvin R. Fremling

Winona State College

Win on a, Minnesota

ABSTRACT

A recycling test apparatus and burrow-containing artificial substrates 

were used to determine the toxicity of the lampricide 3-trifluoromethyl4-nitrophenol (TFM) against Hexagenia mayfly nymphs. Toxicity was 

relatively independent of temperature, but was greater in soft water than 

in hard water, and much greater at low than at high pH's; 12-h LCSO's 

were 4.0 at pH 6.5 and 270.0 at pH 9.5.

INTRODUCTION

Hexagenia mayflies play a vital ecological 

role in lakes, rivers, and streams (Needham 

et al. 1935, Hunt 1953, Britt 1955, Fremling 

1960, Swanson 1967). Nymphs of Hexagenia 

are important to fish because they convert 

organic detritus, algae, and bacteria into high , quality fish food. The detritus-mayfly-fish food 

chain is short and efficient. Inasmuch as the 

life cycle of Hexagenia lasts at least a year 

in the Great Lakes and in most tributary 

streams, nymphs are available to fish in all 

seasons. Because the nymphs pass through 

many molts, sizes are available to suit the 

needs of most fish species.

Since Hexagenia nymphs prefer silt bottoms 

where they can construct burrows, they usually inhabit the same areas used by lamprey 

ammocoetes. Silted streams, for example, 

provide suitable habitat for both. It is important to determine the effect of lampricides 

on Hexagenia nymphs because they may be 

eradicated in lamprey control areas if the 

lampricides are toxic to them.

Hexagenia nymphs are good test organisms 

because they are easily collected and cultured 

and their large size makes them easy to handle 

and observe. Their tendency to abandon their 

burrows when stressed makes it possible to 

accurately assess early effects of toxicants.

METHODS

Either reconstituted water supplied by the 

Fish Control Laboratory, La Crosse, Wis., 

or water from a 12-m deep sand point well 

was used in the tests. The reconstituted 

water contained 48 mg/1 of NaHCO 3 , 30 mg/1 

of CaSO 4 , 30 mg/1 of MgSO4 , and 2 mg/1 of 

KC1; was slightly alkaline (pH 7.2-7.6); and was 

soft (hardness 40-48 and alkalinity 30-35 as 

mg/1 of CaCO 3 ).

The well water was hard, having a total 

alkalinity of 331, total hardness of 384, and 

calcium hardness of 260 (all as mg/1 of 

CaCO3 ). Resistivity at 25 C was 1,277 ohms 

and pH was 7.42. Chemical constitutents 

(mg/1) included ammonia nitrogen, 0.38; 

nitrite, 0.005; nitrate, 0.05; sulfate, 42.5; 

orthophosphate, 0.05; total iron, 0.28; manganese, <0.05; sodium, 32.5; calcium, 59.0; 

magnesium, 18.3; and potassium, 3.7.

Hexagenia nymphs were used as experimental animals, and cultures were maintained 

in the laboratory according to methods described by Fremling (1967). Relatively large 

nymphs (20-22 mm) were used in all experiments because they were easy to handle and 

observe. Last instar nymphs were not knowingly used because physiological stresses 

involved in transformation to the adult stage 

are atypical and emergence during the experiments was not desired. Although most test 

nymphs were H. bilineata from laboratory
Investigations in Fish Control 58 : Fish and Wildlife Service

cultures, their number was supplemented in 

all experiments by nymphs of H. bilineata and 

H. limbata collected from the Mississippi 

River. Species collected from the river were 

not separated because the nymphs were not in 

their last instar and because undue handling 

was undesirable. Nymphs collected from the 

river were placed in laboratory rearing tanks 

to acclimate for at least 1 wk before they were 

used in tests.

Nymphs were collected from the rearing 

tanks by gently sifting mud through a coarse 

screen; they were then transferred to fresh

water where they acclimated for 6 h before 

being used in tests. A large syringe, filled 

with test water from the appropriate vessel, 

was used to flush the remaining nymphs from 

their burrows to determine if any were dead. 

Each nymph was classified as normal, dead, 

or stressed (as indicated by active swimming, 

rapid gill movements, or loss of equilibrium). 

All tests were conducted in a basement laboratory which had no windows. Overhead 

incandescent lamps provided constant light.

Special glass toxicity test vessels (Fig. 1) 

were assembled with silicone glue. Each

BIOASSAY VESSEL 

AIR SUPPLY

NYMPH IN BURROW 

20-LITER JAR

INTERMITTENT 

SIPHON

- FIBER GLASS 

SCREEN

ARTIFICIAL 

SUBSTRATE

SIPHON

Fig. 1. Recycling bioassay apparatus with a burrow-containing epoxy substrate for use with Hexagenia mayfly

nymphs and nonvolatile test chemicals.
Fremling: Acute Toxicity of TFM to Nymphs of Mayflies (Hexagenia sp.)

vessel contained an epoxy resin substrate, 23 

cm long, 5 cm wide, and 5 cm deep. Each substrate contained 10 burrows constructed as 

described by Fremling and Schoening (1973). 

Constant recirculation of test water through 

each vessel was assured by pumping water 

via an air lift (made of 4 and 6 mm I.D. glass 

tubing) from a 20-liter glass jar through the 

test vessel to another 20-liter glass jar via 

an intermittent siphon. A fiber glass screen 

prevented the escape of test animals. Water 

was constantly returned by a siphon (10 mm 

I.D. glass tubing) from the second jar to the 

first. Recirculation of the test water, which 

was permissible because TFM is relatively 

nonvolatile, made it possible to maintain precise control over toxicant concentrations. 

Accumulation of inhibitory concentrations of 

degradation and metabolic products was unlikely because of the large volume of water 

used (38 liters).

Six two-jar units were placed in each of two 

large water baths so that temperature could 

be accurately controlled. Each series of six 

units included one control unit in which no 

TFM was used and five units which contained 

various concentrations of TFM.

Stock solutions of field grade TFM (35.7%) 

and purified TFM (95%) were made by dissolving the chemical in acetone and diluting it

with water. Stock solution was added in equal 

amounts to both jars of each unit, stirred 

thoroughly, and allowed to circulate between 

the jars until mixing was complete. Concentrations of TFM were monitored before 

nymphs were added and periodically throughout each experiment with a Beckman DB 

spectrophotometer (Olson and Marking 1973).

Waters of various hardnesses and pH's 

were used, as described by Marking (1969) 

and Marking and Dawson (1972), respectively. 

LC50 values and 95% confidence limits for 

each test were determined according to 

methods described by Litchfield and Wilcoxon 

(1949).

RESULTS

Purified and field grade TFM were toxic to 

mayfly nymphs at all temperatures tested 

(17.0 - 26.5 C see Table 1). The change in 

toxicity was usually insignificant (P>0.05) for 

single temperature increments; an exception 

being the increment between 23.5 and 24.4 C 

in hard water at 24-h exposure. The exception 

indicates that TFM is more toxic at the higher 

of the two temperatures. Since the temperature difference is small, however, the data 

may reflect biological variation among groups 

of organisms rather than an influence of 

temperature. Although TFM is toxic in short

Table 1. Toxicity of TFM (based on active ingredient) to Hexagenia mayfly nymphs in waters of different 

hardness and temperature. LC50 values and 95% confidence intervals (in parentheses) are listed as /xl/1 for 

35.7% TFM and as mg/1 for 95% TFM.

Hardness 

(mg/l)

384

384

384

384

44

44

Temp. 

(°0

17

21.8

23.5

24.4

18.2

26.5

Formulation

of Tl-M (%') 

6

35.7

95

95

95 11.2 

(10.0-12.5)

35.7

35.7

Hours of

12

10.5 

(8.47-13.0)

10.5 

(9.38-11.8)

6.50 

(5.21-8.11)

4.70 

(3.96-5.58)

exposure

24

6.50 

(5.23-8.07)

6.00 

(5.18-6.95)

7.00 

(5.79-8.47)

3.50 

(2.28-5.36)

4.75 

(4.20-5.37)

3.50 

(2.96-4.14)

96

3.90

(3.0-5.07)

4.30 

(3.45-5.36)

4.20 

(3.41-5.17)

2.50 

(1.80-3.46)

' 2.18 

(1.73-2.74)
Investigations in Fish Control 58: Fish and Wildlife Service

exposures (12-h or less), LCSO's at these exposures are not much greater than those for 

24-h exposures. Comparisons made over a 

wider temperature range might show greater 

significance.

Water hardness influenced the toxlcity of 

TFM to Hexagenia nymphs. After 24-h exposures, TFM was considerably more toxic 

in soft water than in hard water of similar 

pH (7.1-7.6) and temperature (Table 1). The 

24-h LCSO's were 6.50 and 4.75 M l/l of TFM 

in hard and soft water, respectively, at the 

lower temperatures (17.0 and 18.2 C). At the 

higher temperatures (24.4 and 26.5 C), TFM 

was more toxic in soft than in hard water at 

the 12-h exposure but the difference was nil 

at 24-h.

The toxicity of TFM to Hexagenia mayfly 

nymphs was influenced drastically by the pH 

of water (Table 2). The 24-h LC50 at pH 6.5 

(2.50) was significantly greater (P<0.05) 

than that at pH 7.5 (3.35), and the LC50 at 

pH 8.5 (18.8) was more than 5 times the value 

at pH 7.5 (3.35). The toxicity of TFM to the 

nymphs was lowest at pH 9.5 and the 24-h 

LC50 was almost 70 times greater than that 

value at pH 6.5.

DISCUSSION

TFM is apparently less toxic to some invertebrate animals than it is to lampreys. 

Experiments by Erkkila (1962) revealed that 

concentrations of TFM below 20 Ml/1 caused 

insignificant mortality to isopods, gammarids, 

crayfish, dragonflies, water boatmen, and 

case-building caddis flies; that concentrations 

below 10 i/l/l were harmless to glossiphonid 

leeches, stoneflies, bloodworms and snails; 

and that mortality was significant in Hexagenia 

at 6 Ml/1. Smith (1967) showed that mortality 

of hydras, turbellarians, blackflies, and 

Hexagenia mayflies was almost complete in 

the laboratory when these animals were exposed to TFM at 10 /ul/1 for prolonged periods.

In the present study, TFM was toxic to 

Hexagenia mayflies in ranges similar to 

those reported above. Except at pH of 8.5 or 

over, the 24-h LC50 of TFM was always less 

than 10 /ul/1. At low pH in soft water the 

material was especially toxic. At pH 6.5, for 

example, the 96-h LC50 was 1.18 (Table 2).

Table 2, Toxicity of TFM (35.7%) to Hexagenia mayfly nymphs in soft water 

(40-43 mg/1 total hardness as CaCOaJ at temperatures of 22-23 C and at 

selected pH values. LC50 values and 95% confidence intervals (in parentheses) are listed as M!/! TFM.

PH

Hours of exposure

12 24 48 72 96

6.5

7.5

8.5

9.5

4.00 

(3.47-4.61)

27.3

(22.4-33.0)

270 

(221-329)

2.50 

(2.16-2.90)

3.35 

(2.99-3.76)

18.8 

(16.6-21.4)

174 

(156-194)

1.31 

(1.04-1.65)

2.50 

(2.17-2.87)

13. 01

100 

(87.2-115)

__

2.00 

(1.63-2.46)

64.2 

(49.5-83.3)

1.18 

(0.91-1.53)

5.00 

(3.68-6.80)

60.0 

(45.7-78.8)

 " No confidence interval reported because of insufficient data.
Fremling: Acute Toxicity of TFM to Nymphs of Mayflies (Hexagenia sp.)

Hexagenia nymphs are less sensitive than 

larval lampreys to TFM when both species 

are tested in standard laboratory water. 

Dawson et al. (in press) found the 24-h 

LC99 for ammocoetes to be 0.90 at pH 6.5, 

3.25 at pH 7.5, and 12.0 at pH 8.5. In the 

present study, 24-h LC50 values for Hexagenia

nymphs were 2.50 at pH 6.5, 3.35 at pH 7.5, 

and 18.8 at pH 8.5.

In all tests there was a marked tendency 

for treated nymphs to abandon their burrows 

for varying lengths of time before they actually succumbed. In nature, this behavior 

would, on one hand, increase the vulnerability 

of nymphs to predation; on the other hand, 

however, free-swimming nymphs might swim, 

or be swept by the current, out of the zone of 

lethal TFM concentrations. There is no assurance, however, that nymphs would find 

suitable substrate in an open lake or that they 

would recover from the effects of the toxicant.

Hexagenia mayflies are able to recolonize 

denuded areas by downstream drift of nymphs 

and by upstream flight of ovipositing adults 

(Fremling 1973). It is likely that Hexagenia 

populations killed by TFM applications would 

become reestablished. Complete reestablishment would probably require a year or more, 

however.

CONCLUSIONS

1. A recycling toxicity test apparatus with 

artificial substrates was suitable for 

tests of TFM against Hexagenia nymphs.

2. Toxicity of TFM to Hexagenia nymphs is 

relatively independent of temperature.

3. Toxicity of TFM to Hexagenia nymphs is 

greater in soft than in hard water.

4. Toxicity of TFM to Hexagenia nymphs is 

much greater at low than at high pH's.

5. Although TFM is more toxic to ammocetes 

than to Hexagenia nymphs in soft water, 

applications of TFM that exceed the minimum effective concentrations for lamprey 

larvae may kill the nymphs.

ACKNOWLEDGMENTS

I thank Leif L. Marking and other personnel 

of the La Crosse Fish Control Laboratory for 

their advice and assistance throughout this 

study, and Paul A. Boeckman for making most 

of the chemical analyses and many of the 

laboratory observations.

REFERENCES

The artificial substrate apparatus used in 

this study proved very satisfactory as indicated by the fact that in 10 96-h tests the controls showed no mortality in three tests, 10% 

in five tests, 20% in one test, and 30% in one 

test. In the test in which mortality was 30%, 

two of the dead were nymphs which died during transformation to the subimaginal stage. 

Nymphs frequently molted to the next nymphal 

instar in the bioassay vessels.

Artificial substrates such as those used in 

this study provide semidarkness, thigmotactic surfaces, and seclusion for test nymphs. 

Consequently, the nymphs swim less and their 

susceptibility to toxicants is not enhanced by 

fatigue as it is in standard test vessels.

Applegate, V.C., J. H. Howell. Jc W. Moffett, Bc G. H. 

Johnson and M. A. Smith. 1961 C Use of 3-trifluormethyl-4-nitrophenol as a selective sea lamprey larvicide. Great Lakes Fish.Comm.. Tech. Rep. 1. 35 pp.

Britt, N. W. 1955. Hexagenia (Ephemeroptera) population recovery in western Lake Erie following the 1953 

catastrophe. Ecology 36 (3):520-522.

Dawson. V. K.t K. B. Cummings, and P. A. Gilderhus. 

1975. Laboratory efficacy of 3-trifluoromethyl-4- 

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

ServM fewest. Fish Control 63.

Erkkila, L. F. 1962. Lamprey control and research in 

the United States. Pages 25-39 in Great Lakes Fish. 

Comm. Annu. Rep. for 1962.

Fremling, C. R. 1960. Biology of a large mayfly, Hexagenia bilineata (Say), of the Upper Mississippi River. 

Iowa State University, Agricultural and Home Economics Experiment Station, Res. BullM No. 482. pp. 842- 

852.
8 Investigations in Fish Control 58: Fish and Wildlife Service

Fremling, C. R. 1967. Methods for mass-rearing 

Hexagenia mayflies (Ephemeroptera: Ephemeridae). 

Trans. Am. Fish. Soc.. 96(4):407-410.

Fremling, C. R. 1973. Environmental synchronization 

of mass Hexagenia bilineata (Ephemeroptera) emergences from the Mississippi River. Int. Ver. Theor. 

Angew. Limnol. Verh. 18 (part 3):1521-1526.

Fremling, C. R., and G. L. Schoening. 1973. Artificial 

substrates for Hexagenia mayfly nymphs. Pages 209- 

211 in 1st Int. Conf. Ephemeroptera, 1970. Florida 

A & M Univ., Tallahassee, Fla.

Hunt, B. P. 1953. The life history and economic importance of a burrowing mayfly, Hexagenia limbata, 

in southern Michigan lakes. Mich. Dep. Conserv., 

Bull. Inst. Fish. Res., No. 4, 151 pp.

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

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

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

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

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

of biological activity of antimycin determined by fish 

bioassay. Trans. Am. Fish. Soc. 10l(l):100-105.

Needham, J. G., J. R. Traver, and Yin-Chi Hsu. 1935. 

The biology of mayflies. Comstock Publishing Co., 

Ithaca, New York. 759 pp.

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

TFM (lampricide) to six early life stages of rainbow 

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

30(8): 1047-1052.

Smith, B. R. 1967. Lamprey control and research in 

the United States. Pages 28-45 jn Great Lakes Fish. 

Comm. Annu. Rep. for 1965.

Swanson, G. A. 1967. Factors influencing the distribution and abundance of Hexagenia nymphs (Ephemeroptera) in a Missouri River reservoir. Ecology. 

48(2):216-255.
INVESTIGATIONS IN FISH CONTROL

59. Toxicity and Residue Dynamics of the Lampricide 

3-Trif luoromethyl-4-nitrophenol (TFM) in Aquatic 

Invertebrates

By Herman O. Sanders and David F. Walsh

United States Department of the Interior

Fish and Wildlife Service

Washington, D.C. 1975
CONTENTS

Page

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

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

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

Test animals .................................. 4

Toxicity tests ................................. 4

Uptake method ................................. 4

Dissipation method .............................. 5

Reproduction studies............................. 5

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

Acute toxicity. ................................. 5

Chronic toxicity ................................ 5

Uptake study .................................. 5

Dissipation study ............................... 7

Reproduction study .............................. 7

Discussion ...................................... 7

Summary. ....................................... 8

Acknowledgments .................................. 9

Literature Cited. .................................. 9
TOXICITY AND RESIDUE DYNAMICS OF THE LAMPRICIDE

3-TRIFLUOROMETHYL-4-NITROPHENOL (TFM) IN AQUATIC

INVERTEBRATES

By Herman O. Sanders and David F. Walsh 

Fish-Pesticide Research Laboratory, Columbia, Missouri

ABSTRACT

Six species of aquatic invertebrates including scud, Gammarus pseudolimnaeus, daphnid, Daphnia magna, crayfish, Orconectes nais, aquatic 

sowbug, Asellus brevicaudus, damselfly nymph, Ischnura verticalis, and a 

mayfly nymph, Stenonema sp. were exposed to TFM in toxicity tests in 

hard water at 21 C.

The 96-h LC50 of field grade TFM (35.7%) was 57.0 mg/1 for scud and 

110 mg/1 for crayfish; purified TFM (95.7%) was twice as toxic. The 

thirty-day LC50 of field grade TFM was 14 mg/1 for scud and 20 mg/1 for 

crayfish. LC50 values are based on whole formulation rather than active 

ingredient.

14 Uniformly C-ring labeled TFM was employed in the accumulation experiments. All organisms accumulated TFM concentrations within 7 days 

that were up to 58 times (wet weight of whole organism) the concentration in 

water. The biological half-life of TFM in scud was 3.5 days. No reproductive 

impairment occurred in daphnids exposed to 2.4, 4.9, and 10 mg/1 field grade 

TFM for three generations (63 days). Reproduction stopped, however, within 

the first generation when exposed to 18 mg/1.

INTRODUCTION

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) was registered in 1964 for limited use in tributaries of the Great Lakes for 

control of the parasitic sea lamprey (Petromyzon marinus). In 1970, however, the registration of TFM was cancelled by the Environmental Protection Agency because of 

insufficient information on its residues and 

impact on aquatic organisms other than 

lamprey. Since cancellation, extensions have 

been granted to provide time for the Great 

Lakes Fishery Commission to generate the 

necessary information for registration.

The physical and chemical properties, 

efficacy, and toxicity of TFM to aquatic 

organisms have been compiled in an excellent 

review by Schnick (1972). This review is extensive and indicates that only a few studies

report on the toxicology of TFM in aquatic 

invertebrates. Laboratory and field studies 

have shown that TFM is not acutely toxic to 

most fish (Applegate and King 1962; Applegate et al. 1961) and invertebrates (Erkkila 

1962; Smith 1967) when used at concentrations that kill lamprey larvae (Applegate et al. 

1958).

Laboratory studies with TFM and invertebrates have been concerned primarily with 

acute toxicity. Since accumulation and biological effects of TFM residues in aquatic invertebrates are not understood, we initiated 

this study to determine toxicity, uptake, dissipation, and residue magnification of TFM in 

invertebrates. In addition, we evaluated the 

effects of TFM on reproduction in daphnids, 

Daphnia magna. The data in this report are 

intended to assist in the registration of TFM 

and in establishing criteria for permissible
4 Investigations in Fish Control 59: Fish and Wildlife Service

TFM concentrations in the aquatic environment.

MATERIALS AND METHODS

Test Animals

Test animals consisted of four species of 

Crustaceans and two species of early instar 

aquatic insects: mature scud (Gammarus 

pseudolimnaeus Bousfield); early instar and 

mature daphnids (Daphnia magna Strauss); 

14-day-old crayfish (Orconectes nais Faxon); 

mature aquatic sowbug (Asellus brevicaudus 

Forbes); damselfly nymph (Ischnura verticalis 

Say); 'and mayfly nymph (Stenonema sp.). 

Daphnids were from laboratory cultures; all 

other invertebrates were collected from 

streams and ponds near the Fish- Pesticide 

Research Laboratory, Columbia, Missouri. 

Invertebrates collected in the field were 

placed in acclimation tanks for at least 7 days 

before testing.

The water used for cultures and all experiments was from a deep well and had the following characteristics; pH 7.2-7.4 and total 

hardness 270 mg/1 as

Toxicity Tests

The acute toxicity of field grade TFM 

(35.7% active ingredient) and purified TFM 

(95.7% active ingredient) was determined by 

the standard 96-h static toxicity test (Sanders 

1970) and LC50 values were calculated on the 

basis of the total formulation. Thirty-day 

flow-through tests with field-grade TFM were 

conducted using a flow-through diluter after 

Mount and Brungs (1967). In the flow-through 

tests, scud were fed coarsely chopped maple 

and elm leaves and crayfish were fed enriched 

fish-food pellets. All tests were conducted at 

21 ± 1°C.

Toxic effects were measured in terms 

of the median lethal concentration (LC50), 

the toxicant concentration in water which is 

lethal to 50% of the test animals under the 

test conditions. In flow-through tests, the incipient LC50 or lethal threshold concentration (Sprague 1969) was determined when the 

asymptote had been reached in the toxicity

curve. This value was determined when the 

mortality in each aquarium in any 5-day period 

dropped to 10% of the original number of 

animals. Toxicity estimates (LC50 values) 

and corresponding 95% confidence intervals were determined by the Litchfield 

Wilcoxon method (1949).

Uptake Method

Uptake of TFM from water by the four 

species of crustaceans and two species of 

immature insects was studied at concentrations of 0.013, 0.020, 0.026, and 0.510 mg/1.

Uniformly 14C-ring labeled TFM (specific 

activity 3.66 mCi/mM) was used in the accumulation experiments. A sample of TFM 

examined by direct probe mass spectrometry 

contained 0.02% non-volatile 14C-impurities, 

but no impurities of higher molecular weight 

were observed (Analyst, D. L. Stalling, FishPesticide Research Laboratory, Columbia, 

Mo.).

Stock solutions of 14C-TFM were prepared 

in water and further diluted to desired concentrations in a flow-through system. The 

water in each aquarium was renewed at a rate 

of 120 ml/h. The organisms were exposed in 

two-liter glass aquaria containing one liter of 

well water. The flow-through system was 

operated for at least 24 h prior to addition of 

organisms to allow for concentration equilibrium. The organisms were not fed during the 

accumulation experiments.

Invertebrate samples were taken in triplicate, weighed, and prepared directly for 

radiometric analyses by homogenizing the 

whole organism in a tissue grinder. The 

homogenate was obtained by adding 6 ml of 

Triton X-100®: toluene (2: 3 v/v) emulsifier 

to each sample during grinding (Johnson et al. 

1971). This homogenate was then transferred 

to a scintillation vial with three 3 ml washings of a toluene-fluor mixture (5 g of diphenyloxazole (PPO) in 1 liter of toluene). The 

concentration of TFM in water was monitored 

radiometrically by taking triplicate 1 ml 

samples of aquarium water directly into a 

scintillation vial and then adding 14 ml of 

Triton/toluene-fluor mixture. The radioactivity
Sanders and Walsh: Toxicity and Residue Dynamics of TFM in Aquatic Invertebrates

in the tissue and water samples was measured 

with a Beckman 200-L liquid scintillation 

spectrometer. Residue values and magnification factors (residue concentration in organism/residue concentration in exposure water) 

presented in the text and tables were computed on a whole-body, wet weight basis.

Dissipation Method

Dissipation of TFM residues in scud was 

determined by exposing the organisms to TFM 

until a plateau concentration was reached. 

The scud were then transferred to TFM-free 

flowing water and analyzed periodically to 

measure decline in whole-body residues.

Reproduction Studies

Reproductive studies with daphnids were 

conducted in a flow-through system designed 

for exposing small organisms to constant 

concentrations of a toxicant over an extended 

period. Ten first-instar daphnids, up to 24 h 

old, were placed in duplicate exposure vessels containing 1 liter of water. An aqueous 

stock solution of field grade TFM was prepared and then further diluted with water to 

concentrations of 2.4, 4.9, 10, and 18 mg/1. A 

control was included with each test. Daphnids 

were fed a suspension of yeast and algae in 

sufficient amounts to support a stable population. Reproductive success was assessed by 

counting the offspring produced in each TFM 

concentration after the parent daphnids had 

been exposed for 21 days. At the end of 21 

days, 10 of the young from each concentration 

were placed in new media and the 21-day procedure was repeated.

RESULTS

Acute toxicity

Static 96-h toxicity tests indicated that 

field grade TFM (35.7% AI) and purified 

TFM (95.7% AI) have relatively low acute 

toxicities to scud and crayfish (Table 1). The 

96-h LC50 value of field grade TFM was 57 

mg/1 for scud and 110 mg/1 for crayfish. The 

96-h LC50 value of purified TFM was 22 mg/1 

for scud and 55 mg/1 for crayfish. The toxicity to both animals appears to be related to 

the level of active ingredient of the compound.

Chronic toxicity

Thirty-day flow-through toxicity tests of 

field grade TFM with scud resulted in progressively lower LC50 values ranging from 

43 mg/1 at 5 days to 14 mg/1 at 30 days 

(Table2). The incipientLCSO was 14 mg/1 

which was attained in 20 days. The LC50 

values for crayfish exposed to field grade 

TFM declined from greater than 100 mg/1 at 

1 day to 20 mg/1 at 30 days. The incipient 

LC50 for crayfish was 20 mg/1.

The LC50 values for 4-day exposures of 

scud to field grade TFM were similar in 

static and flow-through tests. However, crayfish exposed for 4 days in the flow-through 

tests were resistant to TFM concentrations 

twice as high as those in the static test.

Uptake Study

All invertebrates exposed continuously to 

sublethal concentrations of 14C-TFM accumulated radioactive residues in 7 days that 

were up to 58 times the concentration in water 

(Table 3). After an initial rapid uptake, most 

invertebrates accumulated TFM at a slow rate 

until a plateau was reached at 7 days. Daphnids, 

however, accumulated plateau concentrations 

after a 1-day exposure.

Accumulation of TFM residues by invertebrates appears dependent upon the concentration in water, but magnification factors are 

relatively independent of these concentrations. 

After a 7-day exposure to 0.013 mg/1, scud 

concentrated TFM 58 times (0.754 mg/kg)the 

level in water. When scud were exposed to 

0.510 mg/1, they accumulated total body concentrations 56 times (28.6 mg/kg) that of 

water.

A comparison of the results from our accumulation experiments indicates a significant difference in the rate of uptake and residue magnification of TFM by the various 

organisms. TFM uptake from water by aquatic 

insects was relatively low when compared to 

uptake by Crustacea. However, of all the organisms investigated, crayfish accumulated 

the least TFM concentrations from water.
6 Investigations in Fish Control 59: Fish and Wildlife Service

Table 1. Toxicity1 of field grade TM and purified TM to scud and crayfish

LC50 values 2 (mg/1) and 95$ 

confidence intervals at 

Organism

Scud (mature ) 

Gammarus pseudolimnaeus . .

Crayfish (14 days old)

f^T** c* fvn o n \ o a *n Q ~i a

24

Field grade

100

(83-130) 

130

(115-150)

h

Purified

28

(23-34) 

60

(45-80)

96 h

Field grade

57

(47-69) 

110

(90-125)

Purified

22

(16-31) 

55

(48-70)

 " Static toxicity test. Hard water (pH, 7.2-7.4, total hardness 270 mg/1 as CaCOj

at 21° C.

2Values based on whole formulation rather than active ingredient.

Table 2. Flow-through toxicity tests 1 of field grade TFM against scud

and crayfish

Organism

LC50 values 2 (mg/1) and 95% 

confidence intervals at 

1 day 4 days 10 days 15 days 20 days 30 days

Scud (mature)

Gammarus pseudolimnaeus. >100 43 30 28 14 14

(29-57) (19-48) (17-44) (11-23) (11-23)

Crayfish (14 days old)

Orconectes nais......... >100 34 20 20 20 20

(21-47) (12-32) (12-32) (12-32) (12-32)

water (pH, 7.2-7.4; total hardness 270 mg/1 as CaC03 ) at 21 C. 

2Values based on whole formulation rather than active ingredient.
Sanders and Walsh; Toxicity and Residue Dynamics of TFM in Aquatic Invertebrates 7

Table 3. Uptake and magnification of 14C-TFM by six aquatic invertebrates

Organism

Organisms 1

per

sample

Water2 

concentration 

(mg/1)

Whole "body residues (mg/kg) 

and magnification factor3

1 

day

4 

days

7 

days

14 

days

21 

days

Scud (mature).............. 3 0.013 0.390 0.728 0.754 0.754

Gammarus pseudolimnaeus (30) (56) (58) (58)

Scud (mature).............. 3 0.026 0.286 0.702 1.35 1.33 1.35

Gammarus pseudolimnaeus (11) (27) (51) (51) (52)

Scud (mature).............. 3 0.510 8.45 22.1 28.6 45.7

Gammarus pseudolimnaeus (16) (43) (56) (89)

Waterflea (mature)......... 60 0.026 0.120 0.136

Daphnia magna (5) (5)

Mayfly (early instar)...... 6 0.026 0.0360 0.056 0.110

Stenonema sp_. (1.3) (2.2) (4.4)

Damselfly (early instar)... 3 0.510 0.15 0.63 0.49

Ischnura verticalis (0) (1.2) (l)

Crayfish (immature, 21

days)..................... 2 0.026 0.0360 0.048 0.052 0.068 0.068

Orconectes nais (1.3) (1.8) (2) (2.6) (2.6)

Sowbug (mature)............ 4 0.020 0.28 0.74

Asellus brevicaudus (14) (37)

 " Samples were taken in triplicate.

2Hard water (pH 7.2 and total hardness 270 mg/1 as CaC03 ) at 21 C.

3Concentration in organism (wet weights/concentration in water.

Dissipation study

The dissipation of TFM residues by scud 

was determined by exposing them to 0.026 

mg/1 of 14C-TFM for 7 days. This exposure 

was sufficient to induce a residue plateau. 

Once this plateau was reached, the scuds were 

transferred to TFM-free flowing water. The 

time required for 50% elimination of 

TFM by scud was 3.5 days. At 14 days, 

98% of the radioactive residues had been 

lost. Analytical techniques to determine TFM 

degradation products in invertebrates are not 

well defined but it is assumed that the loss of 

radioactivity was due to excretion of TFM 

and/or metabolites of TFM.

Reproduction study

Continuous exposure of daphnids for three 

generations (63 days) to 2.4, 4.9, and 10 mg/1 

of field grade TFM (35.7% active ingredient) 

did not significantly impair reproduction when 

compared with controls. However, daphnids 

exposed at 18 mg/1 of field grade TFM formed 

ephippial eggs (fertilized eggs) and reproduction stopped within the first generation (21 

days).

DISCUSSION

Of the various chemicals toxic to sea 

lamprey, TFM is considered the most desirable for use in Great Lakes tributaries
8 Investigations in Fish Control 59: Fish and Wildlife Service

because of its effectiveness as a lamprey 

larvicide and its safety to resident fish populations. Concentrations of 2-4 mg/1 of TFM 

required to kill lamprey larvae in streams 

(Applegate et al. 1958) are not toxic to most 

aquatic invertebrates during acute exposures 

(Smith 1967; Erkkila 1962). Observations in 

the field have also shown that fish and reptiles 

(Applegate et al. 1961), and amphibians 

(Johnson 1959) are not affected by these TFM 

treatments.

Comparable data on the toxicities of TFM 

to aquatic invertebrates are limited to a few 

animals because many investigators have not 

included information on water quality or grade 

of TFM used in their experiments. Our LC50 

values for scud and crayfish are in agreement 

with those of Erkkila (1962) and Smith (1967) 

who reported 24-h LC50 values of greater than 

20 mg/1 for both invertebrates. Applegate \ 

et al. (1958) found that the toxic effects of 

mononitrophenols on fish were considerably 

less under conditions that simulated treatment of an actual stream than effects which 

were observed under static conditions. Similar observations were noted in our studies, in 

which TFM was twice as toxic to crayfish in 

static toxicity tests than in flow-through tests.

Toxic chemicals introduced into the aquatic 

environment are often below levels acutely 

toxic to invertebrates. These sublethal concentrations, however, may impair successful 

growth, molting, and reproduction in invertebrate populations. Therefore, populations of 

fishes may be threatened because of loss of 

the fish-food organisms. The results from our 

reproductive toxicity tests indicate no reproductive impairment in D. magna when they were 

exposed continuously for three successive 

generations (63 days) to 10 mg/1 of field grade 

TFM. Because of material cost, the continuous application of TFM in the aquatic environment never exceeds 24 h (Applegate and King 

1962). Therefore, it seems highly improbable 

that daphnids would ever be exposed to concentrations greater than 10 mg/1, especially 

for 63 consecutive days.

Our knowledge of the metabolic fate of TFM 

in fish and invertebrates is incomplete. Lech 

(1971) found that TFM was degraded in rats to

3-trifluoromethyl-4-aminophenol (RTFM). He 

also found that TFM and RTFM were excreted 

in the urine as polar derivatives, some of 

which appear to be glucuronides. Although we 

only determined the loss of 14C-TFM residues in scud, our results suggest that the dissipation of these residues was rapid. Therefore, significant TFM concentrations would 

not be expected to accumulate in top level 

consumers. Further studies, however, are 

needed to determine residue data from various components of simulated or natural intact 

food chains. The method of application, proper 

formulation, water quality characteristics, 

arid species of animals in the area of TFM 

application are important factors to be considered in minimizing the hazard of TFM to 

aquatic animals.

SUMMARY

The acute toxicities of field grade TFM 

(35.7%) and purified TFM (95.7%) were 

determined for scud and crayfish in well 

water (pH 7.2 and total hardness 270 

mg/1) at 21 C. The 96-h LC50 of field grade 

TFM was 57 mg/1 for scud and 110 mg/1 for 

crayfish; purified TFM was twice as toxic. 

The 30-day LC50 of field grade TFM was 

14.3 mg/1 for scud and 20.1 mg/1 for crayfish.

Six species of aquatic invertebrates exposed to 14C-TFM accumulated residues up 

to 58 times, (wet weight) the concentration in 

water.

The amount of TFM accumulated by scud at 

equilibrium (7 days) was proportional to the 

concentration in water. However, magnification factors were relatively independent of 

water concentrations. When scud were transferred to TFM-free flowing water after 7 days 

of exposure to 0.026 mg/1 of TFM, the residues decreased at 14 days from 0.754 mg/kg 

to 0.03 mg/kg.

Concentrations of 2.4, 4.9, and 10 mg/1 of 

field grade TFM did not significantly impair 

reproduction in daphnids after 63 days of exposure. Total production of young was inhibited in 21 days at a concentration of 18 mg/1.
Sanders and Walsh: Toxicity and Residue Dynamics of TFM in Aquatic Invertebrates 9 

ACKNOWLEDGMENTS

This study was partially supported by funds 

from the Great Lakes Fishery Commission, 

Ann Arbor, Mich., and the Fish Control Laboratory, La Crosse, Wis. Assistance in radiometric analysis by James Johnson was greatly 

appreciated.

Johnson, B. T., C. R. Saunders, and H. O. Sanders. 

1971. Biological magnification and degradation of 

DOT and aldrin by freshwater invertebrates. 

Journal Fisheries Research Board of Canada, 

28:705-709.

Lech, J. L. 1971. Metabolism of 3-trifluoromethyl4-nitrophenol (TFM) in the rat. Toxicology and Applied Pharmacology, 20(2): 216-226.

LITERATURE CITED

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

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

larvicide. Great Lakes Fishery Commission, Technical Report, No0 1:1-35.

Applegate, V. CM J. 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-trifluoromethyl-4-nitrophenol 

(TFM) to larval lampreys and eleven species of 

fishes. Trans. Am. Fish. Soci., 91(4):342-345.

Erkkila, L. F0 1962. Lamprey control and research 

in the United States. Pages 25-39, in Great Lakes 

Fishery Commission, Annual Report for the year 

1962, Ann Arbor, Michigan.

Johnson, B. G. H. 1959. Treatment of Pancake River 

(S-56) and Gimlet Creek with Lamprecid 2770, 

August 26 and 27, 1958. Fisheries Research Board 

of Canada, London, Ontario, mimeo. 20 pp. plus 

Tables 1-26.

Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. Journal of Pharmacology and Experimental 

Therapeutics, 96:99-113.

Mount, D. LM and W. A. Brungs. 1967. A device for 

continuous treatment of fish in holding chambers. 

Water Research, 1, 21-29.

Sanders, H, O. 1970. Toxicities of some herbicides to 

six species of freshwater crustaceans. Journal 

Water Pollution Control Federation, 42(8):1544- 

1550.

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

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

larvicide. U.S. Bur. Sport Fish. Wildl. Invest. 

Fish Control No. 44. 31 pp.

Smith, A. J. 1967. The effect of the lamprey larvicide, 

3-trifluoromethyl-4-nitrophenol, on selected aquatic 

invertebrates. Trans. Am. Fish. Soc., (96(4):410-413.

Sprague, J. B. 1969. Measurement of pollutant toxicity 

to fish-I, Biossay methods for acute toxicity. Water 

Research, 3, 793-821.

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

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

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

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