INVESTIGATIONS IN FISH CONTROL

77. Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM), 

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

98:2 Mixture as Lampricides in Laboratory Studies

78. jToxicity of the Molluscicide Bayer 73 and Residue 

of Bayer 2353 in Aquatic Invertebrates

79. Accumulation, Elimination, and Biotransformation of 

the Lampricide 2',5-Dichloro-4'-nitrosalicylanilide 

by Chironomus tentans

UNITED STATES DEPARTMENT OF THE INTERIOR

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

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

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

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

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

(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.

(Reports 53 through 55 are in one cover.)

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

L. Marking. 1973. 11 pp.

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

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

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

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

12pp.

(Reports 56 through 59 are in one cover.)

56. Toxicity of the Lampricide3-Trifluoromethyl4nitrophenol (TFM) to 10 Species of Algae, by A. 

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

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

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

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

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

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

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

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

77. Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM), 

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

98:2 Mixture as Lampricides in Laboratory Studies

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

78. Toxicity of the Molluscicide Bayer 73 and Residue 

Dynamics of Bayer 2353 in Aquatic Invertebrates

By Herman O. Sanders

79. Accumulation, Elimination, and Biotransformation of 

the Lampricide 2',5-Dichloro-4'-nitrosalicylanilide 

by Chironomus tentans

By Joseph A. Kawatski and Ann E. Zittel

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE 

Washington, B.C. 1977
Efficacy of 3-Trifluoromethyl-4-nitrophenol (TFM), 

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

98:2 Mixture as Lampricides in Laboratory Studies

by

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

Fish Control Laboratories, P.O. Box 862

La Crosse, Wisconsin 54601

Abstract

The lampricidal effects of 3-trifluoromethyl-4-nitrophenol (TFM), 2',5-dichloro-4'- 

nitrosalicylanilide (Bayer 73), and a 98:2 mixture of the two (TFM:2B) were tested against 

larvae of the sea lamprey (Petromyzon marinus) under controlled laboratory conditions. The 

lampricides were tested in water at temperatures of 7,12, and 17 C; total hardnesses of 44,170, 

and 300 mg/1 as CaCOs; and pH's of 6.5, 7.5, and 8.5. Temperature had little influence on the 

toxicity of the lampricides, but the effect of Bayer 73 was slowed in cold water. Water hardness 

did not significantly influence the activity of the 98:2 mixture. The toxicities of TFM, Bayer 73, 

and TFM:2B were significantly reduced in water of high pH. Burrowed sea lamprey larvae 

were less vulnerable to TFM, Bayer 73, and TFM:2B than were free-swimming larvae. TFM and 

TFM:2B were selective for free-swimming lampreys over the nontarget organisms used for 

comparison, but the margin of safety for nontarget organisms over burrowed sea lampreys was

Although a number of methods have been used to 

control the parasitic sea lamprey (Petromyzon 

marinus) in the Great Lakes, the most widely used 

and most successful method has been application of 

chemical lampricides. Applegate et al. (1958) reported 

that 3-trifluoromethyl-4-nitrophenol (TFM) was selective against the sea lamprey, and in 1964 the 

compound was registered by the Pesticide Registration Division of the U.S. Department of Agriculture 

for limited use to control sea lamprey larvae in 

tributaries of the Great Lakes.

In 1963, 2',5-dichloro-4'-nitrosalicylanilide (Bayer 

73 or Bayluscide) was found to be extremely toxic to 

sea lampreys, and the addition of small amounts of 

Bayer 73 to TFM greatly reduced the amount of TFM 

required for effective treatment of lamprey populations (Howell et al. 1964). Because Bayer 73 is also 

very toxic to other fish and is virtually nonselective 

for lampreys over rainbow trout (Salmo gairdneri), 

not more than 3% by weight can be added to TFM 

without losing the selective toxicity of TFM (Howell 

et al. 1964). Since 1963, mixtures of TFM and Bayer 73 

have been used to control lampreys in tributaries of 

the Great Lakes by both the United States and 

Canadian governments (Hamilton 1974).

Use of the mixture has produced occasional fish 

kills that could not be predicted on the basis of 

pretreatment toxicity tests (U.S. Bureau of Commercial Fisheries 1968). These fish kills have been

attributed to problems with formulation, application, 

water chemistry, or a combination of these factors 

(U.S. Bureau of Commercial Fisheries 1964, 1968; 

Smith 1966). Numerous tests on the lampricidal 

activity of the combination have been conducted in 

waters of various hardnesses, pH's, and 

temperatures. However, most of the studies were 

conducted in water from natural sources, where the 

presence of several variables and undetermined 

factors made it difficult to evaluate the influence of 

individual water characteristics (Erkkila 1964; 

Howell et al. 1964; U.S. Bureau of Commercial 

Fisheries 1964; Davis et al. 1965; Smith 1966). Recent 

studies supporting continued registration of the 

lampricide mixture have defined the influence of 

water chemistry on the toxicity to nontarget aquatic 

organisms (Kawatski et al. 1974; Kawatski et al. 1975; 

Bills and Marking 1976). Additional information on 

the efficacy of the mixture is needed to support its 

continued registration as a lampricide.

The purpose of the present study was to determine: (1) the toxicities of TFM, Bayer 73, and the 

TFM:2B mixture to sea lamprey larvae; (2) the 

individual and combined influences of temperature, 

water hardness, and pH on toxicity; (3) the toxicities 

to burrowed and exposed (free-swimming) lamprey 

larvae; and (4) the safety to nontarget organisms 

(selectivity).
Materials and Methods

Lamprey larvae collected from the Jordan River 

(Michigan) with electrofishing gear with anesthetized with MS-222 (100 mg/1) and sorted according to 

species and size. Sea lamprey larvae used as test 

organisms (average length, 8 cm; range, 5-10 cm) 

were held for at least 2 weeks before testing in troughs 

containing 12 C well water flowing over a 10-cm-deep 

sand substrate.

Field grade TFM (35.7% in dimethylformamide) 

was obtained from American Hoechst Corporation, 

and Bayer 73 (70% wettable powder) from Chemagro 

Corporation. Bayer 73 was applied at concentrations 

corresponding to 2% of the TFM concentrations, 

based on active ingredients of each, as recommended 

by Howell et al. (1964). The chemicals were tested 

simultaneously, both singly and in combination, at 

each temperature, hardness, and pH. The effect of 

combining the two chemicals was evaluated by the 

use of an additive index (Marking and Dawson 1975).

The toxicants were added to the test vessels 20 h 

after the introduction of lampreys. Ten lamprey 

larvae were exposed to each concentration in 15-liter 

glass jars according to the method of Lennon and 

Walker (1964). Test waters of different quality were 

produced by adding selected reconstituting salts to 

deionized water. The pH in the various tests was 

adjusted and maintained with chemical buffers, as 

suggested by Dawson et al. (1975). Water 

temperatures of 7, 12, and 17 C were controlled by 

water baths.

Dead larvae were counted and removed at 1, 3, 6, 

and 12 h, and daily thereafter, during the 96-h tests. 

Observations are reported at 12 h because that period 

approximates the average duration of chemical 

treatments of streams. LC50 's, LC0 i's, LC99 's, and 

95% confidence intervals were computed according to 

the method of Litchfield and Wilcoxon (1949). The 

LCgg's computed for lampreys statistically approximate the minimum concentrations needed for 

complete kills of the test organisms. Concentrations 

of both toxicants were reported on the basis of active 

ingredient. A P value of 0.05 was used to evaluate 

significance.

Because larvae usually live in burrows in the 

substrate of streams, the toxicity of TFM to burro wed 

lampreys, as well as lampreys confined without 

substrate, was determined. To minimize the effect of 

adsorption of the chemical by the substrate, we 

conducted tests in a flow-through test apparatus 

similar to that used by Marking et al. (1975). 

Burrowed and free-swimming sea lamprey larvae 

were held in separate screened compartments in the 

same test vessel. In addition to the sea lamprey

larvae, we held rainbow trout, brook trout (Salvelinus 

fontinalis), and crayfish (Procambarus sp.) in the test 

chambers during the flow-through toxicity tests to 

accurately assess the selectivity of the lampricides. 

Selectivity was defined by a safety index (LC50 for 

brook trout divided by LCso for sea lampreys) and a 

maximum safety index (LCoi for brook trout divided 

by LCgg for sea lampreys) similar to those employed 

by Marking (1967).

Results 

Effect of Temperature

The 12-h LC 99 's of each compound TFM, Bayer 

73, or TFM:2B against sea lamprey larvae differed 

little at different temperatures (7,12, and 17 C; Table 

1). Temperature did not significantly influence the 

toxicity of any of these compounds at exposure 

periods ranging from 3 to 96 h (Appendix 1), with one 

exception: the activity of Bayer 73 was slightly 

reduced in cold water (7 C) after 3 h of exposure, but 

not after 6 h or longer.

Effect of Water Hardness

Water hardnesses of 44, 170, and 300 mg/1 as 

CaCOs did not significantly influence the toxicity of 

TFM, Bayer 73, or the mixture (Table 1), regardless of 

the period of exposure (Appendix 1).

Effect ofpH

The toxicity of TFM was significantly decreased by 

increases in pH, as indicated by the 12-h LCgg's 

(mg/1) of 0.660 at pH 6.5, 1.70 at 7.5, and 4.65 at 8.5 

(Table 1). Although not as pronounced, the toxicity of 

Bayer 73 also was decreased at high pH's; the 12-h 

LC99 's at pH 6.5, 7.5, and 8.5 were 0.0828, 0.145, and 

0.165 mg/1, respectively (Table 1). Thus Bayer 73 was 

about twice as toxic and TFM about seven times as 

toxic at pH 6.5 as at 8.5. This decreased biological 

activity at high pH's was also evident in data 

reported as LCso's at all exposure periods (Appendix 

1). As expected, the activity of the TFM:2B combination was also reduced at high pH (Table 1).

Effect of Substrate

The lampricide TFM was less toxic to sea lampreys 

burrowed in sand than to larvae confined without a 

substrate. A concentration of 5.63 mg/1 killed all freeswimming larvae but none of the burrowed larvae in 

6 h. In a concentration of 1.83 mg/1, all of the freeswimming larvae, but none of the burrowed larvae,
Table 1. Toxicity (LC99 and 95% confidence interval) 61 of TFM (35.7%), Bayer 73 (70%), and TFM:2B (and 

additive indices for the mixture) to 8-cm sea lamprey larvae after 12 h of exposure in waters of selected 

temperatures, hardnesses, and pH's.

Temp

7

12

17

12

12

12

12

Water hardness 

(mg/lasCaCO 3 )

44

44

44

170

300

44

44

pH

7.5

7.5

7.5

7.5

7.5

6.5

8.5

Individual

TFM

1.60 

1.24-2.07

1.70 

1.11-2.59

1.20 

0.835-1.72

1.59 

1.01-2.51

1.58 

1.00-2.49

0.660 

0.387-1.13

4.65 

3.02-7.15

Bayer 73

0.0680 

0.0407-0.114

0.145 

0.0886-0.237

0.120 

0.0754-0.191

0.125 

0.0741-0.211

0.108 

0.0614-0.190

0.0828 

0.0487-0.141

0.165

0.0971-0.280

Mixture

TFM

1.14 

0.694-1.87

1.80 

1.12-2.90

1.22 

0.782-1.90

1.46 

0.945-2.26

1.48 

0.989-2.22

0.425 

0.277-0.652

3.74 

2.47-5.65

Bayer 73

0.0233 

0.0142-0.0383

0.0367 

0.0231-0.0582

0.0249 

0.0159-0.0391

0.0298 

0.0193-0.0459

0.0302 

0.0203-0.0449

0.00867 

0.00565-0.0133

0.0763 

0.0505-0.115

Additive index 

and range

-0.0551 

-1.45 to 1.17

-0.312 

-2.27 to 0.887

-0.224 

-1.79 to 0.859

-0.157 

-1.86 to 1.14

-0.216 

-1.95 to 0.984

0.336 

-0.958 to 2.51

-0.267 

-2.06 to 0.902

Concentrations based on mg/1 of active ingredient.

Table 2. Toxicity (12-h LC9s for sea lampreys and 12-h LC0 i for rainbow trout, brook trout, and crayfish, and 

95% confidence interval)0- of TFM (35.7%), Bayer 73 (70%), and TFM:2B (and the additive indices for the 

mixture) in flow-through toxicity tests in carbon filtered city water at 12 C.

Species

Sea lamprey 

(burrowed)

Sea lamprey 

(free-swimming)

Rainbow trout

Brook trout

Crayfish

Individual

TFM

5.39 

3.80-7.64

3.00 

2.11-4.26

3.95 

3.43-4.55

4.00 

3.48-4.60

8.20 

7.06-9.53

Bayer 73

0.280 

0.249-0.314

0.0920 

0.0662-0.128

0.0255 

0.0228-0.0286

0.0245 

0.0218-0.0275

>0.150b

Mixture

TFM

12.5 

7.51-20.8

1.64 

1.03-2.61

1.83 

1.63-2.05

2.10 

1.83-2.40

>7.00

Bayer 73

0.255 

0.153-0.425

0.0335 

0.0211-0.0533

0.0374 

0.0333-0.0420

0.0428 

0.0373-0.0491

>0.143

Additive index 

and range

-2.23 

-6.18 to -0.470

0.0979 

-1.04 to 1.46

-0.930 

-1.44 to -0.523

-1.27 

-1.94 to -0.754

Concentrations based on mg/1 of active ingredient. 

No mortality at highest concentration tested.
were dead after 12 h, and 20% of the burrowed larvae 

were still alive after 96 h. The 12-h LC99 's (mg/1) for 

TFM were 5.39 against burrowed and 3.00 against 

free-swimming sea lamprey (Table 2).

In comparison with burrowed lampreys, freeswimming larvae were about three times more 

vulnerable to Bayer 73 and more than seven times 

more vulnerable to TFM:2B. The greater sensitivity 

of free-swimming larvae was evident for each 

chemical individually and in combination, at all 

exposure periods tested (Appendix 2).

Safety to Nontarget 

Organisms (Selectivity)

On the basis of the 12-h LC50 's (Appendix 2), freeswimming (1.88 mg/1) and burrowed (3.39 mg/1) sea 

lamprey larvae were less resistant to TFM than were 

rainbow trout (6.10 mg/1), brook trout (6.00 mg/1), or 

crayfish (12.9 mg/1). However, to show ideal selectivity the lampricide should kill all lamprey larvae 

without harming nontarget organisms. A comparison of the LC 99 's for sea lampreys and the LC Oi 's 

for nontarget species showed TFM to have a rather 

narrow margin of safety. The 12-h LC99 's for 

burrowed (5.39 mg/1) and free-swimming (3.00 mg/1) 

sea lampreys were not significantly different from 

the 12-h LC OJ 's for rainbow trout (3.95 mg/1) or brook 

trout (4.00 mg/1). The crayfish (8.20 mg/1), however, 

were significantly more resistant than freeswimming lampreys (Table 2).

The selectivity of the lampricides can also be 

represented by a safety index (Marking 1967), in 

which a value greater than 1.0 indicates selectivity 

for the target species, and a value less than 1.0 

indicates that nontarget species could be harmed by 

concentrations effective against target species. The

safety index (LC 50 for trout divided by LC50 for 

lampreys) of 1.77 for TFM indicates selectivity for sea 

lampreys; however, the maximum safety index (LC 01 

for trout divided by LC9g for lampreys) of 0.742 

indicates that some mortality of sensitive fishes could 

be expected (Table 3). On the basis of the safety 

indices, Bayer 73 (0.188) and the TFM:2B combination (0.952) did not demonstrate selectivity for 

burrowed sea lampreys in these tests. However, the 

maximum safety indices comparing exposures of 

free-swimming sea lampreys and brook trout to TFM 

(1.33) and TFM:2B (1.28) do show selectivity.

Discussion

The toxicities of TFM, computed on the basis of 

active ingredient, did not differ between the sea 

lamprey larvae used in the present study, which were 

collected in 1973 from the Jordan River (Michigan), 

and those used in a previous study (Dawson et al. 

1975), which were collected in 1972 from the watershed of the Rifle River (Michigan).

Ho well et al. (1964) interpreted the activity of a 

mixture of TFM and Bayer 73 as synergistic if all the 

sea lamprey larvae were killed at concentrations 

which were nontoxic when the chemicals were 

applied singly. Bills and Marking (1976) reported the 

toxicity of the mixture to be additive or less than 

additive (not synergistic) when it was tested against 

fish. The additive indices computed from our data 

support the conclusion that the toxicity of the mixture 

is additive or less than additive. However, Howell et 

al. (1964) and Smith et al. (1974) demonstrated an 

economic advantage of applying the mixture, i.e., the 

amount of TFM required to produce toxicosis was 

reduced while the selectivity was maintained.

Table 3. Safety and maximum safety indices of TFM (35.7%), Bayer 73 (70%), and TFM:2B in flow-through 

toxicity tests against fingerling brook trout and burrowed sea lamprey larvae in carbon filtered city water 

at 12 C.

Chemical

TFM

Bayer 73

TFM:2B

Sea lamprey

12-h LC.-,o

3.39

2.87-4.01

0.180

0.114-0.285

3.15

2.27-4.37

12-h LC9H

5.39

3.80-7.64

0.280

0.249-0.314

12.5

7.51-20.8

Brook trout

12-h LC50

6.00

5.21-6.91

0.0338

0.0301-0.0379

3.00

2.63-3.42

12-h LCoi

4.00

3.48-4.60

0.0245

0.0218-0.0275

2.10

1.83-2.40

Safety index 

and range a

1.77

1.29-2.41

0.188

0.106-0.332

0.952

0.602-1.51

Maximum safety 

index and range b

0.742

0.455-1.21

0.0875

0.0694-0.110

0.168

0.0880-0.320

LC 50 for brook trout/LC 50 for sea lamprey. 

LC01 for brook trout/LC 99 for sea lamprey.
Although temperature changes have been blamed 

for incomplete kills during stream treatments (U.S. 

Bureau of Commercial Fisheries 1958; Smith and 

King 1970), laboratory studies have indicated that 

temperature has little effect on the toxicity of TFM 

(U.S. Bureau of Commercial Fisheries 1960; 

Applegate et al. 1961; Dawson et al. 1975). However, 

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

slowed as the temperature decreased and that the 

selectivity against lampreys increased as the 

temperature dropped to near freezing.

Lowering the temperature has been reported to 

reduce the activity of Bayer 73 (Strufe and Gb'nnert 

1962; Tibbies 1967). Our study indicated reduced 

activity of this compound in cold water after 3 h of 

exposure, but not after longer exposures. Apparently 

the effective contact time is extended at low 

temperatures. Generally, the influence of 

temperature on the lampricides is insignificant when 

compared with influences of other factors.

The reduced activity of TFM at high pH's 

presumably results from an increased ionization of 

the molecule (pKa = 6.07; Applegate et al. 1961). The 

un-ionized form of certain molecules is lipid soluble, 

and therefore more easily transported across the gills 

offish (Sills and Alien 1971). The activity of Bayer 73 

was slightly reduced at higher pH's. This reduction, 

which is consistent with results from previous studies 

(Gillett and Bruaux 1962; Marking and Hogan 1967; 

Farringer 1972), may result from ionization of the 

molecule at higher pH's. Meredith (1971) reported 

some loss of activity of Bayer 73 at pH's below 7, due 

to precipitation. We did not observe this phenomenon, 

probably because of the extremely low concentrations 

used in the toxicity tests.

We found that free-swimming lampreys were more 

vulnerable than burrowed lampreys to TFM, Bayer 

73, and TFM:2B. Possibly the free-swimming lampreys are more excited and have a faster rate of 

metabolism and uptake than the burrowed lampreys. 

Also, the burrowed lampreys may be somewhat 

protected from exposure to the lampricides in the 

water. Field use concentrations of the lampricides for 

each stream are routinely determined in on-site 

toxicity tests against free-swimming lampreys. 

Results of these tests could indicate treatment 

concentrations which are insufficient to eliminate all 

burrowed lampreys. Our results do not support those 

of Applegate et al. (1958), who reported that concentrations of TFM lethal to all larval lampreys were 

essentially the same in jar tests and in treatments of a 

simulated stream.

A comparison of the LC 99 's for sea lampreys and 

the LCoi 's for nontarget species in our tests showed 

TFM to have a narrow margin of safety (working

range). However, Howell and Marquette (1962) 

showed that the working range (minimum lethal to 

maximum allowable concentration) varied from time 

to time in a particular stream and that optimum 

conditions and time for stream treatment could be 

determined by conducting a number of bioassaysin a 

stream.

Conclusions

1. The TFM:2B combination was effective for controlling sea lamprey larvae.

2. Temperature had little influence on the toxicity of 

TFM or TFM:2B to lampreys.

3. The rate of action of Bayer 73 was only slightly 

reduced at low temperatures.

4. Water hardness did not significantly influence the 

activity of the TFM:2B combination.

5. The toxicities of TFM, Bayer 73, andTFM:2B were 

significantly reduced in water of high pH.

6. Burrowed sea lamprey larvae were less vulnerable 

than free-swimming sea lamprey larvae to TFM, 

Bayer 73, and TFM:2B.

7. TFM and TFM:2B are selective for free-swimming 

sea lamprey larvae, but the margin of safety for 

sensitive nontarget organisms over burrowed sea 

lampreys is comparatively narrow.

References

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

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

Applegate, V. C., J. H. Howell, and M. A. 

Smith. 1958. Use of mononitrophenols containing 

halogens as selective sea lamprey larvicides. Science 

127(3294):336-338.

Bills, T. D., and L. L. Marking. 1976. Toxicity of 3-trifluoromethyl-4-nitrophenol (TFM), 2',5-dichloro-4'- 

nitrosalicylanilide (Bayer 73), and a 98:2 mixture to 

fingerlings of seven fish species and to eggs and fry of 

coho salmon. U.S. Fish Wildl. Serv. Invest. Fish Control 

69. 9 pp.

Dodge. 1965. Comparison of bio-assay results using 

TFM and TFM plus Bayer 73,1964. Appendix 12, pages 

67-69 in Great Lakes Fishery Commission, Report of 

annual meeting, Ann Arbor, Michigan, June 22-24,1965.

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

Gilderhus. 1975. Laboratory efficacy of 3-trifluoromethyl-4-nitrophenol (TFM) as a lampricide. U.S. Fish 

Wildl. Serv. Invest. Fish Control 63. 13 pp.

Erkkila, L. F. 1964. Lamprey control and research in the 

United States. Pages 30-41 in Great Lakes Fishery 

Commission annual report for the year 1963. Ann Arbor, 

Michigan.

Farringer, J. E. 1972. The determination of the acute 

toxicity of rotenone and Bayer 73 to selected aquatic 

organisms. M.S. Thesis. University of Wisconsin, La 

Crosse, 32 pp.
Gillett, J., and P. Bruaux. 1962. Laboratory and field 

testing of Bayluscide (Bayer 73). Pflanzenschutz-Nachr. 

15:70-74.

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

of TFM-Bayluscide in fisheries. U.S. Fish Wildl. Serv. 

Lit. Rev. 74-03. NTIS [Natl. Tech. Inf. Serv.] No. PB-235 

442/AS. 39 pp.

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

Hanson. 1964. Synergism of 5,2'-dichloro-4'-nitrosalicylanilide and 3-trifluormethyl-4-nitrophenol in a selective lamprey larvicide. Great Lakes Fish. Comm., Tech. 

Rep. 8. 21 pp.

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

bioassay equipment in the chemical control of sea 

lamprey. U.S. Fish Wildl. Serv. Spec. Sci. Rep. Fish. 418. 

9pp.

Kawatski, J. A., V. K. Dawson, and M. L. 

Reuvers. 1974. Effect of TFM and Bayer 73 on in vivo 

oxygen consumption of the aquatic midge Chironomus 

tentans. Trans. Am. Fish. Soc. 103(3):551-556.

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

Jr. 1975. Acute toxicities of 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide 

(Bayer 73) to larvae of the midge Chironomus tentans. 

U.S. Fish Wildl. Serv. Invest. Fish Control 57. 7 pp.

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

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

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

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

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

Marking, L. L. 1967. Toxicity of MS-222 to selected 

fishes. U.S. Fish Wildl. Serv. Invest. Fish Control 12. 10 

pp.

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

Jr. 1975. Toxicity of the lampricide 3-trifluoromethyl4-nitrophenol (TFM) to nontarget fish in flow-through 

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

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

assessment of toxicity or efficacy of mixtures of 

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

8pp.

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

73 to fish. U.S. Fish Wildl. Serv. Invest. Fish Control 19 

(Resour. Publ. 36). 13 pp.

Meredith, R. 1971. The dependence on pH of the 

molluscicidal activity of the ethanolamine salt of 5,2'-dichloro-4'-nitrosalicylic anilide in solution in 10° artificial 

hard water. WHO [World Health Organ.] 

SCHISTO/71.12-WHO/VCB/71.297. 11 pp.

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

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

Soc. 100(3):544-545.

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

United States. Pages 31-45 in Great Lakes Fishery 

Commission annual report for the year 1964. Ann Arbor, 

Michigan.

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

the United States. Appendix C, pages 29-40 in Great 

Lakes Fishery Commission annual report for the year 

1969. Ann Arbor, Michigan.

Smith, B. R., J. J. Tibbies, and B. G. H. Johnson. 1974. Control of the sea lamprey (Petromyzon 

marinus) in Lake Superior, 1953-70. Great Lakes Fish. 

Comm., Tech. Rep. 26. 60 pp.

Strufe, R., and R. Gonnert. 1962. Comparative studies on 

the influence of environmental factors upon the efficiency of Bayluscide. Pflanzenschutz-Nachr. 15:50-70.

Tibbies, J. J. 1967. Annual report of the Department of 

Fisheries of Canada to the Great Lakes Fishery Commission. Appendix 4, pages 1-111 in Great Lakes Fishery 

Commission, Report of annual meeting, Ann Arbor, 

Michigan, June 20-22, 1967.

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

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

Great Lakes Fishery Commission annual report for the 

year 1958. Ann Arbor, Michigan.

U.S. Bureau of Commercial Fisheries. 1960. Sealamprey 

control and research in the United States in 1960. 

Appendix 4, pages 1-9 in Great Lakes Fishery Commission, Report of annual meeting, Cleveland, Ohio, 

December 1-2, 1960.

U.S. Bureau of Commercial Fisheries. 1964. Progress 

report on sea lamprey control. Appendix 5, pages 1-7 in 

Great Lakes Fishery Commission, Report of annual 

meeting, Ann Arbor, Michigan, June 17-18, 1964.

U.S. Bureau of Commercial Fisheries. 1968. Sealamprey 

program, 1967. Appendix 4, pages 1-32 in Great Lakes 

Fishery Commission, Report of annual meeting, Toronto, Ontario, June 18-20, 1968.
Appendix 1. Toxicity (LC50 and 95% confidence interval) of TFM (35.7%), Bayer 73 (70%), and TFM:2B (and 

additive indices for the mixture) against 8-cm larval sea lampreys in static toxicity test waters of selected 

temperatures, hardnesses", andpH's.

Exposure 

time (h)

Water temp

3

6

12

24

48

96

Water temp

3

6

12

24

48

96

Water temp

3

6

12

24

48

96

Individual

TFM

7 C, hardness 44, 

2.60 

1.97-3.43

1.40 

1.13-1.73

0.800 

0.743-0.861

0.770 

0.637-0.931

0.510 

0.394-0.660

0.345 

0.246-0.484

12 C, hardness 44 

>1.6 C

1.66

1.11-2.50

0.900 

0.708-1.15

0.730 

0.612-0.870

0.730 

0.612-0.870

0.730 

0.612-0.870

17 C, hardness 44 

1.20 

0.880-1.64

0.850 

0.687-1.05

0.740 

0.618-0.885

0.560 

0.452-0.693

0.508 

0.423-0.610

0.496 

0.415-0.593

Bayer 73

pH7.5

0.0970 

0.0765-0.123

0.0550 

0.0393-0.0771

0.0370 

0.0266-0.0515

0.0276 

0.0208-0.0366

0.0220 

0.0158-0.0306

0.0220 

0.0158-0.0306

, pH 7.5 

0.0500 

0.0340-0.0740

0.0500 

0.0340-0.0740

0.0450 

0.0330-0.0608

0.0450 

0.0330-0.0608

0.0450 

0.0330-0.0608

0.0400 

0.0268-0.0598

, pH 7.5 

0.045 

0.0333-0.0608

0.0450 

0.0330-0.0608

0.0410 

0.0309-0.0540

0.0410 

0.0309-0.0540

0.0410 

0.0309-0.0540

0.0410 

0.0309-0.0540

Mixture

TFM

1.05 

0.814-1.35

1.00 

0.745-1.34

0.490 

0.358-0.671

0.455 

0.347-0.597

0.455 

0.347-0.597

0.440 

0.345-0.561

1.00

0.787-1.27

1.00 

0.787-1.27

0.820 

0.610-1.10

0.540 

0.407-0.717

0.450 

0.347-0.584

0.375 

0.255-0.551

0.833 

0.621-1.12

0.485 

0.351-0.670

0.455 

0.350-0.591

0.455 

0.350-0.591

0.455 

0.350-0.591

0.455 

0.350-0.591

Bayer 73

0.0214 

0.0166-0.0275

0.0204 

0.0152-0.0273

0.0100 

0.00730-0.0137

0.00928 

0.00708-0.0122

0.00928 

0.00708-0.0122

0.00898 

0.00704-0.0114

0.0204 

0.0161-0.0259

0.0204 

0.0161-0.0259

0.0164 

0.0124-0.0225

0.0110 

0.00830-0.0146

0.00918 

0.00708-0.0119

0.00765 

0.00520-0.0112

0.0170 

0.0127-0.0228

0.00990 

0.00720-0.0137

0.00928 

0.00710-0.0121

0.00928 

0.00715-0.0121

0.00928 

0.00715-0.0121

0.00928 

0.00715-0.0121

Additive index 

and range

0.601 

-0.0448 to 1.69

-0.0852 

-0.881 to 0.592

0.133 

-0.418 to 0.794

0.0786 

-0.524 to 0.766

-0.314 

-1.29 to 0.321

-0.684 

-2.00 to 0.0606

> -0.0330

-0.0104 

-0.912 to 0.876

-0.276 

-1.24 to 0.357

0.0161 

-0.614 to 0.655

0.219 

-0.315 to 0.941

0.419 

-0.318 to 1.63

-0.0719 

-0.962 to 0.700

0.265 

-0.390 to 1.21

0.189 

-0.348 to 0.898

-0.0388 

-0.699 to 0.571

-0.122 

-0.789 to 0.416

-0.144 

-0.816 to 0.384
Appendix 1. Continued

Exposure

time (h)

Water temp 12 

3

6

12

24

48

96

Water temp 12 

3

6

12

24

48

96

Water temp 12

3

6

12

24

48

96

Individual

TFM

C, hardness 

1.69 

1.33-2.15

1.25 

0.907-1.72

0.770 

0.586-1.01

0.625 

0.488-0.802

0.625 

0.488-0.802

0.625 

0.488-0.802

C, hardness 

2.62 

1.98-3.46

1.54 

1.18-2.02

0.765 

0.582-1.01

0.765 

0.582-1.01

0.710 

0.530-0.951

0.710 

0.530-0.951

C, hardness

0.450 

0.301-0.673

0.300 

0.224-0.401

0.172 

0.121-0.245

0.172 

0.121-0.245

0.172 

0.121-0.245

0.172 

0.121-0.245

Bayer 73

170, pH 7.5 

0.0550 

0.0416-0.0726

0.0380 

0.0259-0.0550

0.0350 

0.0249-0.0491

0.0350 

0.0249-0.0491

0.0350 

0.0249-0.0491

0.0350 

0.0249-0.0491

300, pH 7.5 

0.900 

0.142-5.71

0.0440 

0.0371-0.0603

0.0390 

0.0266-0.0571

0.0390 

0.0266-0.0571

0.0390 

0.0266-0.0571

0.0390 

0.0266-0.0571

44, pH 6.5

0.0480 

0.0360-0.0640

0.0450 

0.0337-0.0602

0.0330 

0.0233-0.0467

0.0310 

0.0227-0.0423

0.0300 

0.0222-0.0406

0.0300 

0.0222-0.0406

Mixture

TFM

1.04 

0.710-1.52

0.630 

0.466-0.852

0.560 

0.435-0.721

0.560 

0.435-0.721

0.560 

0.435-0.721

0.560 

0.435-0.721

1.25 

0.946-1.65

0.860 

0.651-1.14

0.560 

0.449-0.699

0.560 

0.449-0.699

0.560 

0.449-0.699

0.560 

0.449-0.699

0.381 

0.308-0.472

0.232 

0.182-0.295

0.225 

0.176-0.288

0.170 

0.121-0.239

0.170 

0.121-0.239

0.170 

0.121-0.239

Bayer 73

0.0212 

0.0145-0.0310

0.0129 

0.00950-0.0174

0.0114 

0.00888-0.0174

0.0114 

0.00888-0.0147

0.0114 

0.00888-0.0147

0.0114 

0.00888-0.0147

0.0255 

0.0193-0.0340

0.0175 

0.0133-0.0232

0.0114 

0.00920-0.0143

0.0114 

0.00920-0.0143

0.0114 

0.00920-0.0143

0.0114 

0.00920-0.0143

0.00777 

0.00628-0.00960

0.00473 

0.00371-0.00602

0.00459 

0.00359-0.00588

0.00350 

0.00250-0.00490

0.00350 

0.00250-0.00490

0.00350 

0.00250-0.00490

Additive index 

and range

0.00280 

-0.892 to 0.887

0.186 

-0.612 to 1.26

-0.0530 

-0.929 to 0.637

-0.222 

-1.07 to 0.382

-0.222 

-1.07 to 0.382

-0.222 

-1.07 to 0.382

0.979 

-0.0724 to 2.61

0.0458 

-0.591 to 0.840

-0.0243 

-0.739 to 0.645

-0.0243 

-0.739 to 0.645

-0.0810 

-0.856 to 0.579

-0.0810 

-0.856 to 0.579

-0.00850 

-0.835 to 0.799

0.138 

-0.496 to 0.940

-0.447 

-1.63 to 0.257

-0.101 

-1.19 to 0.808

-0.105 

-1.20 to 0.800

-0.105 

-1.20 to 0.800
Appendix 1. Continued

Exposure

time (h)

Water temp 12 

3

6

12

24

48

96

Individual

TFM

C, hardness

>4.00

2.80 

2.26-3.47

2.37 

1.85-3.04

1.40 

1.13-1.74

1.30 

0.957-1.77

1.30 

0.957-1.77

Bayer 73

44, pH 8.5 

0.120 

0.0855-0.168

0.0810 

0.0541-0.121

0.0660 

0.0466-0.0930

0.0440 

0.0310-0.0620

0.0440 

0.0310-0.0620

0.0390 

0.0266-0.0570

Mixture

TFM

3.60 

1.91-6.76

1.26 

1.00-1.59

1.26 

1.00-1.59

1.26 

1.00-1.59

0.580 

0.453-0.743

0.580 

0.453-0.743

Bayer 73

0.0734 

0.0390-0.138

0.0257 

0.0204-0.0324

0.0257 

0.0204-0.0324

0.0257 

0.0204-0.0324

0.0118 

0.00920-0.0152

0.0118 

0.00920-0.0152

Additive index 

and range

>-0.512

0.303 

-0.300 to 1.19

0.0857 

-0.553 to 0.821

-0.484 

-1.45 to 0.104

0.400 

-0.267 to 1.47

0.336 

-0.348 to 1.39

Concentrations based on mg/1 of active ingredient.

Water hardness = mg/1 as CaCO 3 .

No mortality at highest concentration tested.

Appendix 2. Toxicity (LC50 and 95% confidence interval) of TFM(35.7%), Bayer 73 (70%), and TFM:2B (and 

additive indices for the mixture) in flow-through toxicity tests against several aquatic organisms in carbon 

filtered city water at 12 C.

Organism 

and 

exposure 

time (h)

Sea lamprey 

3

6

12

24

48

72

96

Individual

TFM

(burrowed)

>11.9b

9.40 

6.80-13.0

3.39 

2.87-4.01

1.68 

1.45-1.95

1.68 

1.45-1.95

1.68 

1.45-1.95

1.68 

1.45-1.95

Bayer 73

>0.15

>0.15

0.180 

0.114-0.285

0.180 

0.114-0.285

0.180 

0.114-0.285

0.129 

0.0995-0.167

0.129 

0.0995-0.167

Mixture

TFM

>7.80

7.80 

5.32-11.4

3.15 

2.27-4.37

2.35 

1.87-2.95

2.35 

1.87-2.95

2.35 

1.87-2.95

2.35 

1.87-2.95

Bayer 73

>0.159

0.159 

0.109-0.233

0.0643 

0.0463-0.0891

0.0479 

0.0381-0.0602

0.0479 

0.0381-0.0602

0.0479 

0.0381-0.0602

0.0479 

0.0381-0.0602

Additive index 

and range

> -0.890

-0.286 

-1.30 to 0.373

-0.665 

-1.56 to -0.0927

-0.665 

-1.56 to -0.0927

-0.770 

-1.64 to -0.187

-0.770 

-1.64 to -0.187
10

Appendix 2. Continued

Organism 

and 

exposure 

time (h)

Sea lamprey 

3

6

12

24

48

72

96

Brook trout 

3

6

12

24

48

72

96

Individual

TFM

(free-swimming) 

16.6 

12.2-22.5

3.60 

2.98-4.35

1.88 

1.59-2.23

<1.48C

<1.48

<1.48

<1.48

9.65

8.41-11.1

6.00 

4.98-7.23

6.00 

5.21-6.91

6.00 

5.21-6.91

5.95 

5.18-6.83

5.95 

5.18-6.83

5.95 

5.18-6.83

Rainbow trout

3 16.8 

12.3-22.9

6

12

24

48

72

96

6.40 

5.67-7.23

6.10 

5.19-7.18

6.10 

5.19-7.18

6.10 

5.19-7.18

6.10 

5.19-7.18

6.10 

5.19-7.18

Bayer 73

> 0.0800

> 0.0800

0.0625 

0.0540-0.0724

0.0350 

0.0254-0.0482

0.0335 

0.0275-0.0409

0.0335 

0.0275-0.0409

0.0335 

0.0275-0.0409

>0.0800

0.0880 

0.0696-0.111

0.0338 

0.0301-0.0399

0.0338 

0.0300-0.0380

0.0338 

0.0300-0.0380

0.0338 

0.0300-0.0380

0.0338 

0.0300-0.0380

0.0720 

0.0604-0.0858

0.0415 

0.0351-0.0490

0.0353 

0.0315-0.0396

0.0345 

0.0309-0.0386

0.0179 

0.0145-0.0221

0.0179 

0.0145-0.0221

0.0179 

0.0145-0.0221

Mixture

TFM

>7.00

2.31 

2.10-2.54

0.760 

0.573-1.01

0.760 

0.573-1.01

0.700 

0.544-0.900

0.700 

0.544-0.900

0.700 

0.544-0.900

8.10 

6.29-10.4

3.00 

2.63-3.42

3.00 

2.63-3.42

3.00 

2.63-3.42

3.00 

2.63-3.42

3.00 

2.63-3.42

3.00 

2.63-3.42

4.10 

3.57-4.72

3.45 

2.74-4.34

2.47 

2.20-2.77

2.47 

2.20-2.77

2.47 

2.20-2.77

2.47 

2.20-2.77

2.47 

2.20-2.77

Bayer 73

> 0.140

0.0471 

0.0427-0.0518

0.0155 

0.0117-0.0206

0.0155 

0.0117-0.0206

0.0143 

0.0111-0.0184

0.0143 

0.0111-0.0184

0.0143 

0.0111-0.0184

0.165 

0.128-0.212

0.0612 

0.0537-0.0698

0.0612 

0.0537-0.0698

0.0612 

0.0537-0.0698

0.0612 

0.0537-0.0698

0.0612 

0.0537-0.0698

0.0612 

0.0537-0.0698

0.0836 

0.0728-0.0963

0.0704 

0.0559-0.0885

0.0504 

0.0449-0.0565

0.0504 

0.0449-0.0565

0.0504 

0.0449-0.0565

0.0504 

0.0449-0.0565

0.0504 

0.0449-0.0565

Additive index 

and range

> -0.230

0.533 

-0.0222 to 1.39

< 0.0456

<0.111

<0.111

<0.111

>-1.71

-0.195 

-0.690 to 0.180

-1.31 

-1.98 to -0.797

-1.31 

-1.98 to -0.794

-1.31 

-1.99 to -0.798

-1.31 

-1.99 to -0.798

-1.31 

-1.99 to -0.798

-0.405 

-0.978 to -0.00438

-1.24 

-2.29 to -0.520

-0.833 

-1.33 to -0.440

-0.866 

-1.36 to -0.470

-2.22 

-3.43 to -1.34

-2.22 

-3.43 to -1.34

-2.22 

-3.43 to -1.34
11

Appendix 2. Continued

Organism 

and

exposure

time (h)

Crayfish 

3

6

12

24

48

72

96

TFM

>16.5

16.5 

12.2-22.4

12.9 

11.0-15.1

12.9 

11.0-15.1

12.6 

10.5-15.2

12.6 

10.5-15.2

12.6 

10.5-15.2

Individual

Bayer 73

>0.15

>0.15

>0.15

>0.15

>0.15

>0.15

>0.15

TFM

>7.00

>7.00

>7.00

>7.00

>7.00

>7.00

>7.00

Mixture

Bayer 73

>0.140

> 0.140

>0.140

>0.140

>0.140

> 0.140

>0.140

Additive index 

and range

_

Concentrations based on mg/1 of active ingredient.

No mortality at highest concentration tested, 

c

Total mortality at lowest concentration tested.

Toxicity of the Molluscicide Bayer 73 and Residue Dynamics 

of Bayer 2353 in Aquatic Invertebrates

by

Herman O. Sanders

Fish-Pesticide Research Laboratory, Route 1 

Columbia, Missouri 65201

Abstract

The molluscicide Bayer 73 (2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide), a 

chemical used as a synergist in conjunction with 3-trifluoromethyl-4-nitrophenol (TFM) to 

control the sea lamprey (Petromyzon marinus) in tributaries of the Great Lakes, was tested 

against five species of crustaceans and two species of aquatic insects: daphnids (Daphnia 

magna), aquatic sowbugs (Asellus brevicaudus), scuds (Gammarus pseudolimnaeus), glass 

shrimp (Palaemonetes kadiakensis), crayfish (Orconectes nais), damselfly nymphs (Ischnura 

verticalis), and midge larvae (Chironomus plumosus). The acute toxicities ranged from a 48-h 

EC so (median effective concentration causing immobilization) of 0.2 mg/1 for daphnids to a 48- 

h LC 50 (concentration causing 50% mortality) of 25 mg/1 for crayfish. In daphnids exposed 

continuously to Bayer 73, reproduction was not impaired at concentrations of 0.018 and 

0.032 mg/1, but was significantly (P<0.01) reduced at concentrations of 0.056, 0.10, and 

0.18 mg/1. Exposure to Bayer concentrations of 0.56, l.O.and 1.8mg/1 significantly (P<0.01) 

reduced emergency of midges. All organisms exposed to Bayer 2353 (chlorosalicylic acid ring 

UL 14 C) accumulated radioactive residues in 24 h that ranged from 4 to 80 times (based on wet 

weight of whole organism) the water concentration of 1.2 ± 0.2 // g/1. Scuds and midge larvae 

eliminated 90% of the residues in 48 h.

The molluscicide Bayer 73 (2-aminoethanol salt of 

2',5-dichloro-4'-nitrosalicylanilide), which is sold 

commercially as Bayluscide, is especially toxic to 

freshwater snails (Chemagro Corporation 1970). It 

has been used in Africa, southeastern Asia, and 

portions of South America to control several species 

of snails that are intermediate hosts of organisms 

causing schistosomiasis in man (Gb'nnert 1962). 

Bayer 73 is effective as an ovicide against snail eggs 

(Gillet and Bruaux 1962), as a herbicide to control the 

tropical water fern, Saluinia aurticulata (Wild and 

Mitchell 1970), and as a piscicide for controlling fish 

populations (Brynildson 1970). This molluscicide is 

also extremely toxic to larvae of the sea lamprey, 

Petromyzon marinus (Howell et al. 1964), and has 

been used by the U.S. Fish and Wildlife Service and 

the Canadian Department of Environment for 

sampling lamprey populations in tributaries of the 

upper Great Lakes. It is also used as a synergist with 

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

for lamprey control (Howell et al. 1964).

Authorization for lampricidal uses of TFM and the 

TFM-Bayer 73 mixture was jeopardized when the 

U.S. Department of Agriculture, Pesticide Regulation

Division, gave notice on 13 May 1970 that registration of TFM would be cancelled unless tolerances 

were obtained in fish and water. The U.S. Fish and 

Wildlife Service was granted an extension from the 

then new regulatory agency, the U.S. Environmental 

Protection Agency (EPA), on 22 February 1971, to 

obtain additional data on methodology for application, and toxicology of, TFM, Bayer 73, and their 

combination. Submissions for an Amended Registration and Petition for Exemption from Tolerance for 

TFM were filed with EPA in February 1976.

Laboratory and field studies have shown that a 0.1- 

mg/1 concentration of Bayer 73, which is effective for 

controlling lamprey larvae (Howell et al. 1964; Smith 

1967), could have an adverse effect on nontarget 

aquatic organisms, such as planarians, tubificids, 

and daphnids (Hunn 1973), mollusks (Gb'nnert 1962), 

and fishes (Marking and Hogan 1967). However, 

aquatic invertebrates with a hard exoskeleton, such 

as ostracods (Kawatski 1973), aquatic sowbugs, 

crayfish, dragonflies, and dobsonflies (Hunn 1973), 

and stonefly naiads (Sanders and Cope 1968) were 

not severely affected by exposure to Bayer 73. Meyer 

and Howell (1975) reported that nymphs of burrowing

1
mayflies (Hexagenia sp.) exposed to Bayer 73 in Lake 

Huron water at 12 C were 160 times more resistant to 

this chemical than were larval lampreys.

Although the effects of TFM on aquatic invertebrates have been documented (Smith 1967; 

Chandler and Marking 1975; Fremling 1975; 

Kawatski et al. 1975; Sanders and Walsh 1975), a 

recent review of the literature (Hamilton 1974) 

indicated a lack of information for evaluating the 

safety of Bayer 73 to aquatic invertebrates. The 

objectives of the study were to determine the acute 

toxicities of Bayer 73 to aquatic invertebrates in 

static tests and to determine the effect of continuous 

exposure of Bayer 73 on reproduction in daphnids 

(Daphnia magna) and emergence of midges 

(Chironomus plumosus). In addition, the accumulation of 14C-Bayer 2353 from water by seven aquatic 

invertebrates was determined.

Materials and Methods

Test animals included five species of crustaceans 

and two species of aquatic insects: early instar and 

mature daphnids; mature aquatic sowbugs (Asellus 

breuicaudus); mature scud (Gammarus pseudolimnaeus); mature glass shrimp (Palaemonetes 

kadiakensis); juvenile crayfish (Orconectes rials); 

early instar damselfly nymphs (Ischnura verticalis); 

and first and early fourth instars of midge larvae. 

Daphnids, scuds, and midge larvae were from 

laboratory cultures and the other invertebrates were 

collected from streams or ponds near Columbia, 

Missouri. All organisms were acclimated to 

laboratory conditions by rearing or holding them in 

the dilution water at the test temperature. A combination of Duro-test and wide spectrum Grow-lux bulbs 

provided light for the cultures and all tests. The light 

cycle was controlled for a 16-h photoperiod.

The water used for cultures and all experiments 

was from a deep well; it had a pH of 7.4 and a total 

hardness of 270 mg/1 as CaCO3 . Crayfish and midge 

larvae were exposed at 22 ± 1 C and all other 

organisms at 18 ± 1 C.

The Bayer 73 (Chemagro Corp., Lot No. 8059410) 

was supplied by the Fish Control Laboratory, 

LaCrosse, Wisconsin, as a wettable powder containing 70% 2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide. Concentrations were based on 

active ingredient.

Acute toxicity tests were conducted under static 

conditions; methods used were those recommended 

for standardized laboratory toxicity tests (Committee 

on Methods for Toxicity Tests with Aquatic 

Organisms 1975). Flow-through tests were performed 

in a system modeled after Mount and Brungs (1967). 

The method of Litchfield and Wilcoxon (1949) was

used to estimate the LC 50 's (concentrations causing 

50% mortality) or FC50 's (median effective concentrations causing immobilization) and 95% confidence 

intervals. 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.

Reproductive studies were conducted with 

daphnids in a flow-through system, with a chemical 

delivery apparatus designed by Chandler et al. (1974). 

Ten first-instar daphnids up to 24 h old were placed 

in duplicate exposure vessels that contained 1 liter of 

water. Thus, 20 daphnids per group were exposed 

continuously through a complete life cycle (21 days) 

to concentrations of 0 (the control), 0.018,0.032,0.056, 

0.10, and 0.18 mg/1 of Bayer 73. The test solutions 

and controls contained 0.1 ml/1 of ethanol. Test 

organisms were fed a suspension of yeast in sufficient 

amounts to support a stable population. Reproductive 

success was assessed by counting the offspring 

produced in each concentration and the control after 

the parent daphnids had been exposed for 21 days. 

The mean number of young produced per adult was 

determined by averaging the number of young 

produced in replicate tests. Data were analyzed by 

analysis of variance, and significant differences 

among treatments were determined by a multiple 

means comparison test (least significant difference, 

Snedecor and Cochran 1974).

Use of the rearing technique described by Biever 

(1965) for the colonization of chironomid larvae 

maintained a continuously reproducing population of 

midges. Before the experiments were begun, eggs 

collected from a rearing unit were hatched in a 

stender dish. One hundred first-instar larvae 

(1.5 mm long and up to 24 h old) were transferred 

into exposure vessels that had been previously 

prepared by adding 13 g of washed sand to 1 liter of 

water. During the test, larvae were fed a commercial 

dog food called Dog Kisses (Biever 1965).

A flow-through system as described in the daphnid 

studies was used to determine the effects of Bayer 73 

on larval survival, pupation, and emergency of 

midges. Cast pupal skins at the water surface in test 

containers were counted and removed daily to 

determine emergence. The test was terminated at 30 

days, when 80-90% of the control larvae had emerged. 

The effects of Bayer 73 on emergence were analyzed 

by analysis of variance on the arcsin transformation 

for proportions (angle = arcsin/percentage) followed by a least significant difference test (Snedecor and 

Cochran 1974).

The radioactive Bayer 2353 (chlorosalicylic acid 

ring UL 14C) used in this study was prepared by the
American Radiochemical Corporation, Sanford, 

Florida, and was obtained through the Fish Control 

Laboratory, LaCrosse, Wisconsin. The specific activity of 10.0 mCi/mM given for this labeled compound 

was confirmed by gas chromatographic analysis of 

the stock solutions.

The concentration used in the accumulation studies 

(1.2 ± 0.2 /xg/1) was selected partly on the basis of 

the acute toxicity determined for the most sensitive 

species exposed. Johnson and Schoettger (1975) 

suggested that a concentration between 1/10 and 

1/1000 of the EC 50 , depending on the slope of the 

toxicity curve, be selected as the test concentration. 

On the basis of use pattern, field evaluations, and 

toxicity of the chemical, a factor of 0.01 was applied to 

the lowest confidence limit of the EC50 for D. magna.

Accumulation studies were conducted in a flowthrough system that included the chemical delivery 

apparatus designed by Chandler et al. (1974). 

Exposure vessels were 2-liter glass aquaria containing 1 liter of well water. Stock solutions of the 14CBayer 2353 were prepared in water and further 

diluted in the flow-through system. Organisms were 

not fed during the 5-day exposure.

Invertebrate samples were taken in triplicate, 

weighed, and prepared directly for radiometric 

analysis by homogenizing the whole organism. The 

homogenate was obtained by adding 6 ml of Triton 

X-100:toluene (2:3 v/v) emulsifier to each sample 

before grinding it (Johnson et al. 1971). The concentration of labeled compound in water was monitored 

radiometrically by taking triplicate 1-ml samples 

entering the exposure chambers and then adding 

14 ml of Triton :toluene-fluor mixture. The radioactivity of the samples was measured with a Beckman 

200-L liquid scintillation counter. All samples were 

corrected for quench and counted until a statistical 

counting error of 5% or less had been reached. Residue 

values and accumulation factors (residue concentration in organism/residue concentration in exposure 

water) were computed on a whole-body, wet weight 

basis.

Elimination of 14C-Bayer 2353 residues in several of 

the invertebrates was measured by exposing the 

organisms to the compound until a plateau concentration was reached. The organisms were then 

transferred to clean flowing water and analyzed at 8, 

24, and 48 h to measure the decline in whole-body 

residues.

A method proposed by Mount and Stephan (1967) 

for establishing acceptable toxicant limits for aquatic 

organisms under continuous exposure conditions 

was used to calculate an application factor for 

determining a toxicant concentration that has no 

adverse effect on reproduction in daphnids or life

cycle alterations in chironomids. The application 

factor consists of the laboratory determined maximum acceptable toxicant concentration (MATC) 

that has no effect on the test animals during chronic 

exposure, divided by the 48-h EC50 .

Results

Toxicity

The acute toxicities of Bayer 73 varied greatly 

among aquatic invertebrates, and ranged from a 48-h 

EC50 of 0.2 mg/1 for daphnids to a 48-h LC 50 of 

25 mg/1 for crayfish (Table 1). The invertebrates with 

a soft integument (daphnids and midge larvae) were 

more susceptible than those with a highly chitinized 

exoskeleton (scud, glass shrimp, and crayfish).

Flow-through tests indicated that the compound 

was not toxic to early instar crayfish in a concentration of 50 mg/1 for 24 h. However, continuous 

exposure produced toxic effects, and at 4 days the 

LC 50 was 35 mg/1. Toxicity reached an asymptote at 

10 day sand a time-independent LC 50 of 16 mg/1 was 

estimated. The toxicity to scuds was not substantially 

affected by continuous exposure, and 24-h LC50 's 

were similar in static and flow-through tests.

Daphnid Reproduction

Continuous exposure of daphnids through a 

complete life cycle (21 days) to 0.056, 0.10, or 

0.18 mg/1 of Bayer 73 in a flow-through system

Table 1. Acute toxicity of Bayer 73 to five species of 

aquatic invertebrates in static tests.

LC 50 (mg/1) and 

95% confidence limits a

Organism and stage

Daphnid, 1st instar

Scud, mature

Glass shrimp, mature

Crayfish, juvenile

Midge, 4th instar larva

24 h

(5.6)

4.7-6.7

(19)

12-29

(32)

23-45

(2.1)

1.6-2.9

96 h

(0.2)b

0.14-0.27

(2.4)

1.8-3.1

(10)

7-15

(25)

19-33

1.5b

1.1-2.2

a Toxicities are expressed in terms of EC 50 for daphnid and 

midge larva and LC , for the other organisms.

b 48-h values.
Table 2. Survival and reproduction of Daphnia 

magna after a 21-day exposure to Bayer 73 at 

18 ±1 C.

Concentration 

(mg/1)

Percent survival 

of adults a

Total young

produced per

surviving adult

0.0

0.018

0.032

0.056

0.10

0.18

95

95

85

90

85

45

1,586

1,545

1,553

726b

244b

101 b

Twenty young exposed at each concentration.

Significantly different from controls (P<0.01), n = 2.

significantly reduced(P<0.01) reproduction (Table 2). 

Survival of the adult daphnids at the termination of 

the tests was 85-95% in all Bayer 73 concentrations 

and controls, except at 0.18 mg/1, in which adult 

survival was 45%. The number of young produced per 

parent daphnid ranged from 5 at 0.18 mg/1 to 77 at 

concentrations of 0.032 and 0.018 mg/1. The mean 

number of young produced in the controls was 79.

The MATC for daphnids was estimated to be 

between 0.032 and 0.056 mg/1. The application factor 

(MATC/ECso) was between 0.16 and 0.28 (Table 3).

Midge Emergence

Emergence of adult midges was significantly 

reduced (P<0.01) after 30 days exposure to Bayer 73 

at concentrations of 0.56,1.0, and 1.8 mg/1 (Table 4). 

Emergence was significantly delayed (P <0.05) in the 

0.32 mg/1 concentration at 15 days; however, at 30 

days the emergence time was similar to that in the 

controls. The pupal stage seems to be the most

Table 3. Ranges of application factors and safe 

concentrations for Bayer 73, continuous exposure, 

for Daphnia magna and the midge Chironomus 

plumosus.

48 h EC 50 MATCb Application 

Organism (mg/l) a (mg/1) factor 0

Daphnid 0.2 0.032-0.056 0.16-0.28 

(0.14-0.27)

Midge larva 1.5 0.32-0.56 0.21-0.36 

(1.1-2.2)

a Values in parentheses are 95% confidence limits of the

EC 50 .

Highest concentration producing no observed effect and 

the lowest concentration in which an effect was observed.

c Limits of the maximum acceptable toxicant concentration (MATC), divided by 48-h EC50 .

sensitive stage in the midge life cycle; some pupal 

mortality was noted in control chambers. The highest 

pupal mortality was in the 1.8-mg/l concentration; 

only 33% of the organisms emerged.

The MATC for midges was estimated to be between 

0.32 and 0.56 mg/1 and the application factor was 

between 0.21 and 0.36 (Table 3).

Accumulation and Elimination

The accumulation of 14C-Bayer 2353 was relatively 

low in the seven species of aquatic invertebrates, 

ranging from 4 to 80 times the water concentration of 

1.2 ± 0.2 fjg/l (Table 5). Accumulation of radioactive residues was greater in soft-bodied invertebrates 

(daphnids and midge larvae) than in organisms with 

a hard exoskeleton (glass shrimp and crayfish). Most 

invertebrates accumulated maximum residues

Table 4. Cumulative percentages of midges (Chironomus plumosus) that emerged after continuous exposure 

of the larvae to different concentrations of Bayer 73 at 22 ± 1 C.

Days of

exposure

15

20

25

30

Bayer 73 concentration (mg/1)

0

(Control)

10

57

84

87

0.1

5

49

79

85

0.32

3a

47

78

86

0.56

4 a

34b

45b

47b

1.0 1.8

3a O a

15b 5b

24b 27b

53b 33b

a Significantly different from controls (P<0.05). 

Significantly different from controls (P<0.01).
Table 5. Whole-body residues of 14C-Bayer 2353 accumulated from water by seven aquatic invertebrates.

Organism and stage

Daphnid, 1st instar

Aquatic sowbug, mature

Scud, mature

Glass shrimp, mature

Crayfish, juvenile

Damselfly, mature nymph

Midge, 4th instar larva

Water 

concentration 

(^tg/landSE)

1.4 (0.02)

1.1 (0.08)

1.2 (0.25)

1.0 (0.09)

1.0 (0.17)

1.2 (0.10)

1.1 (0.03)

Whole-body residues (^ug/kg) 

and accumulation factors 3

24 h

75(4)

[53]

25(4)

[23]

80 (4)

[67]

4 (2)

[4]

4 (2)

[4]

8(2)

[7]

87 (7)

[80]

48 h

78 (2)

[56]

32 (2)

[29]

88 (2)

[73]

6(2)

[6]

8(2)

[8]

12(1)

[10]

90 (5)

[82]

72 h

80 (2)

[57]

30(1)

[27]

83 (4)

[69]

9 (1)

[9]

9(1)

[9]

10(1)

[8]

89 (2)

[81]

120 h

75(3)

[53]

28(1)

[25]

85 (2)

[71]

9 (1)

[9]

H (1)

[H]

8(2)

[7]

80 (6)

[73]

a r» i -,, , . ,, . , . , ,,

of the concentration in the organism to the concentration in water.

between 24 h and 48 h of continuous exposure and 

changed little after an additional exposure (up to 

120 h).

Residues in crustaceans after a 24-h exposure to 

14C-Bayer 2353 ranged from 4 fi g/kg in glass shrimp 

and crayfish to 80 /x g/kg in scuds. Concentrations of 

residues in scuds declined by 50% within 8 h and by 

90% after 48 h in clean flowing water. Aquatic insects 

exposed continuously accumulated residues in 24 h 

that ranged from 8 /i g/kg for damselfly nymphs to 

87 fi g/kg for midge larvae. Midge larvae eliminated 

50% of the residues in about 11 h and 90% in 48 h.

Discussion

Bayer 73 and TFM are often applied in combination 

as a single application in tributaries of the Great 

Lakes to control larval lampreys. Because of costs 

and the efficacy of the compounds, continuous 

application to streams never exceeds 12 h (Applegate 

and King 1962). Since nontarget organisms would 

generally be exposed to Bayer 73 for only a short time, 

the acute toxicities would seemingly be most important in assessing the likelihood of mortalities of 

aquatic invertebrates within the area of a lampricide 

application; however, some of the chemical may be 

adsorbed and retained by bottom sediments (Strufe 

and Gonnert 1962), and benthic organisms could be 

exposed for a longer time than the duration of the

lampricide treatment. Results of our midge 

emergence studies indicate that there was no effect on 

emergence of midges at a concentration of 0.32 mg/1, 

a concentration double that normally applied in sea 

lamprey control (Howell et al. 1964).

The low accumulation and rapid elimination rate of 

radioactive residues in scuds and midge larvae 

suggest that Bayer 73 would not accumulate through 

the food chain to upper-level consumers. Techniques 

for determination of degradation products of Bayer 

2353 in invertebrates are not well defined, but it is 

assumed that the loss of radioactivity was due to 

excretion and degradation of the parent compound.

Field observations and laboratory tests have 

shown that Bayer 73 is less toxic to most aquatic 

invertebrates than it is to sea lampreys. Smith (1967) 

reported that the granular formulation applied in a 

stream at about three times the normal lampricide 

application rate had little effect on benthic invertebrates. Kawatski (1973) found that an ostracod 

(Cypretta kawatai) would not be adversely affected at 

concentrations used to control sea lampreys. 

Daphnids appear to be more sensitive than most 

other aquatic invertebrates; consequently, a reduction in population could occur during a lamprey 

treatment. Daphnid populations could also be 

reduced if a concentration of 0.1 mg/1 was maintained for 21 days. This duration seems highly 

improbable, since use exposures do not exceed 12 h.
Meyer and Howell (1975) indicated that if a 

theoretical concentration of 0.11 mg/1 were applied 

to the bottom 5 cm of standing water, the concentration would be sufficiently diluted at 10.7 cm above 

the bottom to be harmless to trout. Inasmuch as trout 

are more sensitive to Bayer 73 (Marking and Hogan 

1967) than are most of the crustaceans and aquatic 

insects that have been exposed, concentrations used 

for lamprey control should have little effect on most 

arthropods. Moreover, a mixture (98:2 ratio) of TFM 

and Bayer 73 was more toxic to larval sea lampreys 

than to nontarget fish in comparable laboratory 

toxicity tests (Bills and Marking 1976).

The results from this study and others indicate that 

if standard lamprey control procedures are followed 

and a Bayer 73 concentration of 0.1 mg/1 is not 

exceeded for more than 24 h, no adverse effect on 

most aquatic invertebrates should occur. However, 

data derived from controlled laboratory experiments 

can serve as guidelines for field applications only 

after differences in water quality, species of 

organisms, methods of application, and toxicant 

formulation have been carefully considered.

Conclusions

1. Invertebrates with a soft integument were more 

susceptible to Bayer 73 than were those with a 

chitinized exoskeleton.

2. Daphnid reproduction was significantly reduced 

(P<0.01) in Bayer 73 concentrations of 0.056, 0.10, 

and 0.18 mg/1.

3. Emergence of midges was significantly reduced 

(P<0.01) in Bayer 73 concentrations of 0.56, 1.0 

and 1.8 mg/1.

4. Equilibria of 14C-Bayer 2353 residues were attained in most invertebrates in 24 h, when total 

body residues (wet weight of whole organism) 

ranged from 4 to 80 times the water concentration.

5. Elimination of radioactive residues in scuds and 

midge larvae was rapid: 90% of the accumulated 

residues were lost within 48 h after the organisms 

were transferred to clean flowing water.

6. Concentrations of Bayer 73 that are effective in 

controlling lamprey should have little adverse 

effect on most aquatic invertebrates.

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the sea lamprey, 1973. Pages 61-65 in Great Lakes 

Fishery Commission annual report for the year 1973.

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

continuous treatment of fish in holding chambers. Water 

Res. 1:21-29.

Mount, D. I., and C. E. Stephan. 1967. A method for 

establishing acceptable toxicant limits for fish 

malathion and the butoxyethanol ester of 2,4-D. Trans. 

Am. Fish. Soc. 96(2): 185-193.

Sanders, H. O., and O. B. Cope. 1968. The relative 

toxicities of several pesticides to naiads of three species 

of stoneflies. Limnol. Oceanogr. 13(1):112-117.

Sanders, H. O., and D. F. Walsh. 1975. Toxicity and 

residue dynamics of the lampricide 3-trifluoromethyl-4- 

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

Wildl. Serv. Invest. Fish Control 59. 9 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.

Snedecor, G. W., and W. C. Cochran. 1974. Statistical 

methods. Iowa State Univ. Press, Ames. 327 pp.

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

fish-I. Bioassay methods for acute toxicity. Water Res. 

3:793-821.

Strufe, R., andR. Gonnert. 1962. Comparative studies on 

the influence of environmental factors upon the efficiency of Bayluscide. Pflanzenschutz-Nachr.-Bayer 15:50-70.

Wild, H., and D. S. Mitchell. 1970. The effect of 

Bayluscide on the water fern Saluinia aurticulata and 

other aquatic plants. Pflanzenschutz-Nachr.-Bayer 

23(2):105-110.

Accumulation, Elimination, and Biotransformation of the

Lampricide 2',5-Dichloro-4'-nitrosalicylanilide

by Chironomus tentans1

by

Joseph A. Kawatski and Ann E. Zittel

Department of Biology, Viterbo College

La Crosse, Wisconsin 54601

Abstract

When exposed to sublethal concentrations of 14C-2',5-dichloro-4'-nitrosalicylanilide (Bayer 

2353), either alone or in combination with 3-trifluoromethyl-4-nitrophenol (TFM), larvae of the 

midge Chironomus tentans accumlated 14C residue rapidly. Uptake was related directly to 

amount of toxicant present in exposure water and to water hardness. Residues of 14C-Bayer 

2353 did not attain a stable uptake equilibrium but were rapidly excreted, both during 

continuous exposure and during postexposure periods in toxicant free water. Chironomids 

biotrans formed 14C-Bayer 2353 to H C-chlorosalicylic acid and an unidentified 14C metabolite.

In 1958, salicylanilides, particularly 2',5-dichloro4'-nitrosalicylanilide (Bayer 2353), were discovered to 

be potent molluscicides (Gonnert 1962). After further 

investigation, the U.S. Fish and Wildlife Service and 

the Canadian Department of Environment began in 

1966 to use a 5% granular formulation of Bayer 73 (2- 

aminoethanol salt of Bayer 2353) to control the sea 

lamprey, Petromyzon marinus (Hamilton 1974a, 

1974b). Mixtures of Bayer 73 and 3-trifluoromethyl-4- 

nitrophenol (TFM) in a 2:98 ratio (by weight) have 

also been used because the addition of small amounts 

of Bayer 73 substantially reduces the amount of TFM 

needed for effective treatment of populations of larval 

lampreys (Howell et al. 1964).

The toxicity of Bayer 73 to nontarget invertebrates 

varies widely. Bayer 73, at the concentrations used 

for lamprey control, does not appear to affect many 

hard-shelled aquatic invertebrates (Rye 1972; Gumming and Dawson 1973; Fish-Pesticide Research 

Laboratory 1973; Hunn 1973; and Sanders 1973), but 

some soft-bodied invertebrates are susceptible (Rye 

1972). Additional information regarding the 

dynamics of the toxicant and its residues in nontarget invertebrates is required to satisfy regulatory 

requirements of the U.S. Environmental Protection 

Agency.

The present study was designed to determine the 

rates of accumulation and elimination of Bayer 2353 

by larvae of the aquatic midge Chironomus tentans 

(Diptera; Chironomidae) during short-term sublethal

exposures. In addition, we investigated the ability of 

the organism to metabolize the toxicant.

Materials and Methods

Radioactive Bayer 2353 (2',5-dichloro-4'-nitrosalicylanilide) was synthesized with a uniformly labeled 

14C-chlorosalicylic acid ring (lOmCi/mM, American 

Radiochemical Corporation, Sanford, Florida). Nonradioactive Bayer 73 (2-aminoethanol salt of 2'-5-dichloro-4'-nitrosalicylanilide, 70% wettable powder) 

was obtained from the Chemagro Corporation, 

Kansas City, Kansas. TFM (95.7% 3-trifluoromethyl4-nitrophenol) was used in a 98:2 (by weight) 

combination with Bayer 2353; this mixture is 

designated as TFM-2B. Reconstituted water was 

prepared as described by Marking (1970) to effect 

three levels of hardness (mg/1 as CaCO 3 ) and (in 

parentheses) pH: soft, 40-48 (7.2-7.6); hard, 160-180 

(7.6-8.0); and very hard, 280-320 (8.0-8.4). These 

materials were supplied by the Fish Control 

Laboratory, La Crosse, Wisconsin. All other reagents 

were of analytical grade unless otherwise specified.

Stock cultures of Chironomus tentans, a widely 

distributed midge whose benthic larva is an important fish food organism, were maintained in continuous laboratory culture in soft water at 21 ± 2 C 

according to the procedure of Kawatski et al. (1975). 

Only fourth instar larvae were used in the experiments. Before exposure to the toxicant, the

'This study was funded by Contract 14-16-0008-807, U.S. Fish and Wildlife Service.
animals were removed from the rearing system and 

acclimated in 900 ml of the toxicant free test water at 

20 + 0.5 C for 24 h.

For accumulation studies, larvae were exposed to 

14C-Bayer 2353 in Pyrex beakers containing 900 ml of 

water (60-80 larvae per beaker). Before a sublethal 

concentration of the toxicant (54-108 /Ag/1) was 

added, samples of larvae and test water w-ere 

withdrawn for background radiation determinations. 

The toxicant was added in an acetone-water (1:1) 

solution; the acetone concentration in test water 

never exceeded 1 ml/1. Triplicate samples of larvae 

and test water were employed throughout the study.

Immediately after addition of the toxicant, 0.5-ml 

samples of the exposure water were collected. The rate 

of toxicant accumulation by larvae was determined 

by sampling animals and water at 2-h intervals 

during the first 8 h of exposure, and after 12, 24, 48, 

72, and 96 h of exposure. Three larvae per sample 

were withdrawn from each exposure vessel, placed on 

an absorbent towel, blotted dry, and weighed. Dry 

weights were calculated on the basis of a determined 

dry weight correction factor. Each sample was then 

transferred to a scintillation vial where 0.5 ml NCS 

tissue digestor (Amersham/Searle Corp.) was added. 

The vials were held at room temperature for 2 h 

before the addition of 10 ml of scintillation cocktail 

(4 g of 2,4-diphenyloxozole, 0.10 g of 1,4-bis [2(5- 

phenyloxozolyl)] benzene, 250 ml of Triton X-100, 

and 750 ml of toluene). The vials were shaken 

vigorously and then refrigerated until radioactivity 

of the contents was determined.

In elimination studies, the larvae were transferred 

from the exposure vessels to 900 ml of toxicant free 

water after 24 h of exposure to the toxicant. Samples 

of larvae and water were withdrawn at 2-h intervals 

and prepared for radioactivity determinations by the 

same counting procedure as used in the accumulation 

studies.

Biotransformation studies were conducted by the 

following procedures. About 100 larvae were exposed 

to a sublethal concentration of 14C-Bayer 2353 

(108 /u.g/1). Thirty larvae were withdrawn at 12-h 

intervals over a 36-h period. The larvae were blotted 

dry, transferred to a grinding vessel, and homogenized with a motor-driven Teflon pestle. We added 

small amounts of double distilled water, and then 

subjected the homogenate to ultransonic vibrations, 

using the intermediate tip of a Sonic 300 Dismembrator (Artex Systems Corporation) at 50% of full 

power for 10 min.

The sonicated homogenate was spotted directly 

on precoated thin layer plates purchased from 

Brinkman Instrument Company (Polygram SIL NHR, 0.2 mm) and Eastman Kodak Company (6061 

Silica Gel, 0.1 mm). The developing systems

were: methanol/chloroforrn/ammonium hydroxide 

(50/25/2.5,vol/vol/vol; J. J. Lech, personal communication) and methanol/chloroform/acetic acid 

(16/8/l,vol/vol/vol). For visualization of the separation, we superimposed a mixture of nonradioactive 

Bayer 73 and chlorosalicylic acid on the spotted 

sonicate. We vertically sectioned developed plates 

from 0.5 cm below to 10 cm above the spotting 

points, and by using the standards as horizontal 

reference points, cut the vertical sections into several 

pieces (Kawatski and McDonald 1974). The pieces 

were placed in individual counting vials with 10 ml 

of scintillation cocktail, and their radioactivity was 

determined.

The radioactivity in samples was measured with a 

Nuclear Chicago Mark II scintillation spectrometer. 

Observed counts per minute were converted to 

disintegrations per minute (DPM) with the channels 

ratio-external standard method of quench correction. 

For accumulation and elimination studies, DPM's 

were converted to micrograms of toxicant (in terms of 

the weight of the parent Bayer 2353 molecule) and 

expressed in terms of the weight or volume of the 

sample. In biotransformation studies, the DPM's 

were summed for each vertical section of the thin 

layer plate and the percentages of the total contributed by individual horizontal sections were determined.

Results

The magnitude of 14C-Bayer 2353 accumulation 

from water by Chironomus tentans during continuous sublethal exposure was in part a function of 

the Bayer 2353 concentrations in exposure water. The 

initial rate of uptake was related both to toxicant 

concentration and to hardness of exposure water 

(Fig. 1). For example, when animals were exposed to 

63 /A g/1 of Bayer 2353, uptake rates during the first 

12 h were 12.8, 11.9, and 9.6 /xg/g per hour in soft, 

hard, and very hard water; when the exposure 

concentration was 108 fig/I, the initial uptake rates 

were 46.7,42.0, and 31.3 /xg/g per hour, respectively.

In nearly all uptake experiments where test 

animals were exposed continuously to Bayer 2353 for 

up to 96 h, maximum accumulation occurred during 

the first 24 h, and usually within 12 h. Residue 

accumulation did not plateau; rather, the elimination 

rates thereafter exceeded the uptake rates.

When chironomids that had been exposed to Bayer 

2353 were placed in toxicant free water, 14C residues 

continued to be excreted very rapidly, and the rate of 

excretion was directly proportional to the toxicant 

concentration during exposure and to the maximum 

amount of Bayer 2353 accumulated during the 

exposure (Fig. 2). Biological half-lives (time required
7001

LLJ

EXPOSURE TIME (HOURS)

Fig. 1. Uptake of 1 4C residue (//, g/g dry wt) by Chironomus tentans larvae from water of three hardnesses (0, soft; n, hard; 

very hard) that initially contained 63(A) or 108(B) /j.g/1 of 14C-Bayer 2353.

for loss of one-half of the accumulated material) of the 

residue varied from 3.5 to 25.4 h (Table 1), and the 

rate of elimination was directly proportional to the 

maximum amount of residue accumulated during the 

exposure period (Fig. 3).

Uptake of Bayer 2353 by chironomids exposed to 

TFM-2B was no different from uptake of Bayer 2353 

from test water containing only Bayer 2353 (Table 2). 

When chironomids were exposed to 2.3 /ig/1 of Bayer 

2353 and to TFM-2B (112 /j. g/1 of TFM; 2.3 /tg/lof 

Bayer 2353), they accumulated 14C residue at the rate 

of 0.59 /i g/g per hour during the initial 12 h; 

thereafter, residue was eliminated even during 

continuous exposure.

When various substrates (paper, sand, and silt) 

were placed in exposure vessels, Bayer 2353 concentrations in the water decreased with time at a faster

rate than when substrates were absent. Presumably 

because of the reduction in toxicant concentration, 

chironomids in the vessels containing substrates 

absorbed Bayer 2353 residue less rapidly than did 

control animals in substrate free systems (Table 3). 

Chironomids that absorbed 14C-Bayer 2353 were 

able to cleave the 14C-Bayer 2353 molecule, as 

evidenced by the recovery of 14C labeled chlorosalicylic acid (Table 4). As exposure continued beyond 

8 h, a decreasing percentage of the accumulated 

residue in chironomid tissue was chlorosalicylic acid 

and an increasing percentage was Bayer 2353, 

suggesting that chlorosalicylic acid was being 

excreted. One other 14C metabolite observed, more 

polar than either chlorosalicylic acid or Bayer 2353, 

was probably acidic. A third 14C-labeled component 

appeared at all exposure times; it did not migrate in
300-

LLJ

CO 

CO

O> 

X

CO

200-

100

CQ 

0»

0

12 24/0 12 

TIME (HOURS)

24

Fig. 2. Uptake of 14C residue (/*g/g dry wt) by Chironomus tentans larvae from soft water that initially contained 54 (0) or 

162 (Q) /tg/1 of 14C-Bayer 2353, and elimination of residue during a 24-h postexposure period in toxicant free water.

our thin layer chromatographic systems, however, 

and was not present in the 14C-Bayer 2353 standard.

Discussion

The manner of uptake and elimination of Bayer 

2353 by Chironomus tentans helps to explain the 

toxic effects of Bayer 73 observed by Kawatski et al. 

(1975). In 8-h exposures, Bayer 73 was more toxic in 

soft water than in hard water, but in longer exposures 

(24-96 h) the differences in toxicity related to water 

hardness became progressively less, and were 

statistically nonsignificant. The uptake pattern for

14C-Bayer 2353 is consistent with the toxicity data 

from previous static tests. During the first 8 h of 

exposure, the rate of 14C-Bayer 2353 uptake decreased 

with increasing water hardness, but after about 12 h 

of exposure, residue levels did not vary significantly 

in relation to hardness. Furthermore, after 12 h of 

exposure and during continuous exposure, 14C residue 

was eliminated regardless of water hardness. The 

toxicological result of this excretion is that 48-, 72-, 

and 96-h LC50 values do not differ significantly at 

any given water hardness (Kawatski et al. 1975).

Accumulation of 14C-Bayer 73 is independent of 

TFM uptake; i.e., Bayer 2353 absorption is identical 

from solutions of either TFM-2B or Bayer 2353 alone.
Table 1. Accumulation of 14C residue (pg/g dry wt) by Chironomus tentans larvae at 20±1 C under various 

conditions of exposure to 14 G-Bayer 2353, elimination of 14C residue during postexpoture in toxicant free 

water of differing hardness and pH, and biological half-life of accumulated 14C residue a -

Initial 

exposure concn. 

of Bayer 2353 

(Atg/D a

Experiment 1

54

162

Experiment 2

108

108

Experiment 3

108

108

108

Experiment 4

108

108

108

Experiment 5

108

108

108

Exposure 

water 

hardness"

S

S

H

H

S

S

S

H

H

H

VH

VH

VH

Length of 

exposure 

(h)

24

24

8

12

24

24

24

24

24

24

24

24

24

Total 

14C residue 

accumulated 

(jug/g) c

79

356

171

164

567

567

567

634

634

634

488

488

488

Postexposure 

water 

hardness"

S

S

H

H

S

H

VH

S

H

VH

S

H

VH

Biological 

half-life of 

14C residue 

(h)

25.4

12.6

12.0

10.7

15.2

5.0

3.7

3.5

3.5

3.5

5.2

5.2

4.2

a Results of simultaneous tests are grouped as experiments.

b S = soft (hardness, 40-48 mg/1 as CaCO ;! ; pH, 7.2-7.6); H = hard (160-180 mg/1; pH, 7.6-8.0); and VH = very hard 

(280-320 mg/1; pH, 8.0-8.4).

c Expressed in terms of the weight and activity of the parent Bayer 2353 molecule.

Kawatski and Bittner (1975) found that accumulation 

of TFM was likewise independent of Bayer 73 uptake. 

Therefore the slight synergistic toxicity of TFM and 

Bayer 73 is not due to potentiated uptake at absorptive surfaces but must be due to other interactions. 

The ability of chironomids to metabolize 14C-Bayer 

2353 to form at least two compounds (chlorosalicylic 

acid and a more polar material) probably accounts in 

part for the rapid excretory rate. The unidentified 

polar material is probably a conjugated form of either 

Bayer 2353 itself or of chlorosalicylic acid. Salicylates are readily conjugated with glycine and 

glucuronic acid in higher organisms (Milne 1963), 

and chironomids also possess such detoxication 

systems (Kawatski and Bittner 1975). Since only the 

chlorosalicylic acid ring of the Bayer 2353 was

labeled, the chloronitroaniline ring and possible 

derivatives of it were not recovered or measured.

The H C material that did not migrate on thin layer 

plates (nonmigrating material; NMM) cannot with 

certainty be identified as a Bayer 2353 metabolite, 

since it may represent an artifact of the analytical 

method. If Bayer 2353 or its metabolites are trapped 

within cell fragments which remain immobile, the 

material identified as NMM may simply be Bayer 

2353, chlorosalicylic acid, or the more polar unidentified derivative. Although we attempted to disrupt 

subcellular components by sonicating the tissue 

homogenates before performing thin layer chromatography, some salicylates probably remained bound 

to proteins (a well-known tendency for salicylates). 

Thus the identity of the NMM remains undetermined.
600

O)

x,

O)

[2 400

OL

S 

200

x 

<

0

0 20 40 60 80 

jug BAYER 73 LOST/g TISSUE/h

Fig 3. Relation between rate of elimination of accumulated 1 4C residue and maximum body burden of residue (//, g/g dry wt).

Table 2. Accumulation of 14C residue by Chironomus tentans larvae during 96 h of continuous exposure to 

sublethal concentrations of 14C-Bayer 2353 and TFM-2Ba in soft water (CaCO3 hardness, 40-48 mg/l;pH, 

7.2-7.6) at 20 + 1 Cb .

Exposed to Bayer 2353

Length of 

exposure 

(h)

0 

2 

4 

8 

12 

24 

96

Exposure 

water 

(/*g/l)

2.52 (0.11) 

2".39 (0.23) 

2.14 (0.24) 

1.99 (0.15) 

1.88 (0.05) 

1.73 (0.13) 

1.78 (0.12)

TFM-2B designates a 98:2 (by weight) combination 

Standard deviations are shown in parentheses.

Tissue

(ft g/g 

dry wt)

1.55 (0.41) 

2.32 (0.32) 

4.84 (1.63) 

6.37 (1.04) 

3.72 (1.06) 

1.69 (0.16)

of TFM and Bayer 2353.

Exposed to TFM-2B

Exposure 

water 

(/ig/1)

2.52 (0.12) 

2.29 (0.04) 

2.21 (0.11) 

1.98 (0.19) 

1.97 (0.29) 

1.74 (0.03) 

2.13 (0.06)

Tissue 

(/xg/g 

dry wt)

2.09 (0.15) 

2.49 (0.35) 

4.64 (1.40) 

6.64 (1.38) 

5.20 (0.60) 

1.65 (0.12)
Table 3. Accumulation of 14C residue (fjig/g dry wt) by Chironomus tentans larvae during 36 h of continuous 

exposure to 14C-Bayer 2353 in soft water (CaCO3 hardness, 40-48 mg/l; pH, 7.2-7.6) at 20 + 1 C 

containing 5 g of the named substrates in 900 ml of test mediuma .

Water characteristics at end of exposure

Substrate and

mesh number

(where applicable)

None

Paper

Sand

Silt

toweling

30

80

200

30

80

4

43.3

(8.7)

34.3

(11.6)

36.3

(13.0)

22.7

(12.0)

28.7

(12.5)

23.3

(18.8)

38.2

(15.3)

Duration of exposure (h)

8

121.6

(17.8)

36.1

(1.0)

42.6

(12.3)

23.7

(1.9)

59.2

(13.2)

45.0

(17.4)

95.0

(58.6)

12

99.5

(6.7)

74.1

(13.9)

76.5

(15.9)

23.8

(2.7)

41.2

(3.1)

44.5

(22.0)

50.1

(34.3)

24

155.6

(105.9)

71.0

(22.6)

41.9

(9.1)

30.0

(6.9)

74.0

(22.6)

34.3

(8.8)

65.4

(56.1)

36

185.1

(95.2)

54.3

(13.6)

34.4

(1.1)

24.4

(5.0)

51.4

(8.5)

39.7

(6.4)

26.1

(9.2)

Bayer 2353

(/tg/D

67.1

(1.7)

40.9

(0.8)

60.5

(1.3)

44.8

(0.8)

61.7

(3.4)

44.1

(1.5)

26.4

(1.0)

Hardness

(mg/lasCaCO 3 )

52

53

57

78

70

120

140

Dissolved

oxygen

(mg/l)

7.7

7.3

8.3

6.0

7.3

5.3

5.0

pH

6.8

6.8

6.8

6.9

6.8

7.0

6.8

Initial concentration of 14C-Bayer 2353: 66.0 ju.g/1; standard deviations are shown in parentheses.
Table 4. Percentages of total 14 C residue retained by 

Chironomus tentans larvae exposed continuously 

to 14C-Bayer 2353 in soft water (CaCO3 hardness, 

40-48 mg/l;pH, 7.2-7.6) at 20 + 1 Ca .

Thin layer chromatographic 

identification

Component

Bayer 2353

Chlorosalicylic

acid

Otherb

NMM e

R(»

(ranges in 

parentheses)

0.65

(0.55-0.70)

0.45

(0.30-0.55)

0.25

(0.10-0.30)

0.0

(-0.05-0.10)

Exposure period (h) c

8

20.7

(3.2)

32.5

(4.0)

18.1

(4.3)

28.5

(1.7)

24

23.8

(2.9)

29.9

(4.3)

15.9

(3.1)

29.2

(2.9)

36

39.1 d

(13.1)

23.7 d

(13.0)

8.8 d

(2.4)

26.2

(3.9)

* Initial concentration of 14C-Bayer 2353: 108 /tg/1.

Rf's for both acidic and basic thin layer systems; "other"

material appeared to migrate slightly behind Bayer 2353

in the basic system. 

, Standard deviations given in parentheses.

Change between 8 and 36 h statistically significant

(P = 0.05). 

e NMM: nonmigrating material; the amounts of NMM were

similar in both basic and acidic thin layer developing

systems.

Conclusions

1. Larvae of Chironomus tentans accumulated Bayer 

2353 rapidly from sublethal exposure concentrations.

2. Accumulation of residue depended in part on water 

hardness and concentration of the toxicant.

3. Chironomids eliminated residue rapidly, both 

during continuous exposure and during postexposure periods in toxicant free water.

4. Rates of Bayer 2353 accumulation and elimination 

were independent of TFM absorption and elimination.

5. C. tentans metabolized 14C-Bayer 2353, producing 

14C-chlorosalicylic acid and at least one other 14C 

product.

References

Gumming, K. B., and V. K. Dawson. 1973. Laboratory 

efficacy of lampricides. Presented at Great Lakes 

Fishery Commission Interim Meeting, Ann Arbor, 

Michigan, December 4-5, 1973. 2 pp.

Fish-Pesticide Research Laboratory. 1973. Quarterly 

progress report July-September 1973. U.S. Fish Wildl. 

Serv., Fish-Pest. Res. Lab., Columbia, Missouri. 29 pp.

Gonnert, R. 1962. Bayluscide, a new compound for 

controlling medically important freshwater snails. 

Pflanzenschutz-Nachr. 15:4-24.

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

of Bayluscide in fisheries. U.S. Fish Wildl. Serv. Lit. Rev. 

72-02. Natl. Tech. Inf. Serv. No. PB-235 441/AS. 60 pp.

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

of TFM-Bayluscide in fisheries. U.S. Fish Wildl. Serv. 

Lit. Rev. 74-03. Natl. Tech. Inf. Serv. No. PB-235 442/AS. 

39pp.

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

Hanson. 1964. Synergism of 5,2'-dichloro-4'-nitro-salicylanilide and 3-trifluormethyl-4-nitrophenol in a selective lamprey larvicide. Great Lakes Fish. Comm., Tech. 

Rep. 8. 21 pp.

Hunn, J. B. 1973. Registration-oriented research on TFM 

in 1972. Appendix 22, pages 310-315 in Great Lakes 

Fishery Commission Report of annual meeting, Ottawa, 

Ontario, June 19-21, 1973.

Kawatski, J. A., and M. A. Bittner. 1975. Uptake, 

elimination, and biotransformation of the lampricide 3- 

trifluoromethyl-4-nitrophenol (TFM) by larvae of the 

aquatic midge Chironomus tentans. Toxicology 

4:183-194.

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

Jr. 1975. Acute toxicities of 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4-nitrosalicylanilide 

(Bayer 73) to larvae of the midge Chironomus tentans. 

U.S. Fish Wildl. Serv. Invest. Fish Control No. 57. 7 pp.

Kawatski, J. A., and M. J. McDonald. 1974. Effect of 3-trifluoromethyl-4-nitrophenol on in vivo tissue respiration 

of four species of fish with preliminary notes on its in 

vitro biotransformation. Comp. Gen. Pharmacol. 

5:67-76.

Marking, L. L. 1970. Juglone (5-hydroxy-l,4- 

napthoquinone) as a fish toxicant. Trans. Am. Fish. Soc. 

99(3):510-514.

Milne, M. D. 1963. The excretion of salicylate and its 

metabolites. Pages 18-27 in Salicylates: an international 

symposium. J & A Churchill Ltd., London. 310 pp.

Rye, P. R., Jr. 1972. The toxicity of two lamprey larvicides to selected aquatic invertebrates. U.S. Fish Wildl. 

Serv., Hammond Bay Biological Station, Millersburg, 

Michigan. 14 pp. (Unpubl. manuscr.)

Sanders, H. O. 1973. Toxicity and accumulation of Bayer 

73 in aquatic invertebrates. Presented at Great Lakes 

Fishery Commission Interim Meeting, Ann Arbor, 

Michigan, December 4-5, 1973. 1 p.


(Reports 60 through 62 are in one cover.)

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

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

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

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

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

(Reports 63 through 66 are in one cover.)

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

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

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

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

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

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

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

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

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

V. K. Dawson. 1975. 7 pp.

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

by Ralph M. Burress. 1975. 8 pp.

(Reports 69 and 70 are in one cover.)

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

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

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

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

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

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

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

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

Bills. 1976. 11 pp.

(Reports 73 through 76 are in one cover.)

73. Formalin: Its Toxicity to Nontarget Aquatic Organisms, Persistence, and Counteraction, by T. 

D. Bills, L. L. Marking, and J. H. Chandler, Jr. 1977. 7 pp.

74. Chlorine: Its Toxicity to Fish and Detoxification of Antimycin, by L. L. Marking and T. D. 

Bills. 1977. 6 pp.

75. Malachite Green: Its Toxicity to Aquatic Organisms, Persistence, and Removal with Activated 

Carbon, by T. D. Bills, L. L. Marking, and J. H. Chandler, Jr. 1977. 6 pp.

76. Toxicity of Furanace® to Fish, Aquatic Invertebrates, and Frog Eggs and Larvae, by L. L. 

Marking, T. D. Bills, and J. H. Chandler, Jr. 1977. 6 pp.

Fish Control Laboratories

Fish and Wildlife Service

U.S. Department of the Interior

P.O. Box 862 

La Crosse, Wisconsin 54601
As the Nation's principal conservation agency, the Department of the 

Interior has responsibility for most of our nationally owned public lands 

and natural resources. This includes fostering the wisest use of our land 

and water "resources, protecting our fish and wildlife, preserving the 

environmental and cultural values of our national parks and historical 

places, and providing for the enjoyment of life through outdoor 

recreation. The Department assesses our energy and mineral resources 

and works to assure that their development is in the best interests of all 

our people. The Department also has a major responsibility for American 

Indian reservation communities and for people who live in island 

territories under U.S. administration.

GPO 837 -72E

FISH CONTROL LABORATORIES 

U.S. FISH AND WILDLIFE SERVICE

P.O. BOX 862 

LA CROSSE. WISCONSIN 546O1

POSTAGE AND FEES PAID 

U.S. DEPARTMENT OF THE INTERIOR

INT423