INVESTIGATIONS IN FISH CONTROL

83. Survival of Two Species of Freshwater Clams, 

Corbicula leana and Magnonaias boykiniana, After 

Exposure to Antimycin

84. Chronic and Simulated Use-Pattern Exposures of 

Brook Trout (Salvelinus fontinalis) to 

3-Trifluoromethyl-4-nitrophenol(TFM)

85. Hydrolysis and Photolysis of the Lampricide 

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

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 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. 

8pp.

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. 

9pp.

(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. 1973.12 pp.

(Reports 56 through 59 are in one cover.)

56. Toxicity of the Lampricide 3-Trifluoromethyl-4-nitrophenol (TFM) to 10 Species of 

Algae, by A. A. Maki, L. D. Geissel, and H. E. Johnson. 1975.17 pp.

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

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

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

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

59. Toxicity and Residue Dynamics of the Lampricide 3-Trifluoromethyl-4-nitrophenol 

(TFM) in Aquatic Invertebrates, by H. O. Sanders and D. F. Walsh. 1975.9 pp.

(Reports 60 through 62 are in one cover.)

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

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

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

Flow-Through Tests, by L. L. Marking, T. D. Bills, and J. H. Chandler. 1975.9 pp.

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

83. Survival of Two Species of Freshwater Clams, 

Corbicula leana and Magnonaias boykiniana, After 

Exposure to Antimycin

By L. L. Marking and J. H. Chandler, Jr.

84. Chronic and Simulated Use-Pattern Exposures of 

Brook Trout (Salvelinus fontinalis) to 

3-Trifluoromethyl-4-nitrophenol (TFM)

By W. P. Dwyer, F. L. Mayer, J. L. Alien, sand D. R. Buckler

85. Hydrolysis and Photolysis of the Lampricide 

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

By D. P. Schultz,, and P. D. Harman

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE

Washington, D.C. 1978

Survival of Two Species of Freshwater Clams,

Corbicula leana and Magnonaias boykiniana,

After Exposure to Antimycin

by

L. L. Marking

U.S. Fish and Wildlife Service

Fish Control Laboratory 

La Crosse, Wisconsin 54601

and 

J. H. Chandler, Jr.

U.S. Fish and Wildlife Service

Southeastern Fish Control Laboratory

Warm Springs, Georgia 31830

Abstract

The Asiatic clam, Corbicula leana Prime, and a clam native to the southern United States, 

Magnonaias boykiniana, were exposed to the fish toxicant antimycin at several concentrations 

for various periods and then placed in an untreated earthen pond for posttreatment observation. Both species survived the concentrations and exposure periods usually used in field application. However, latent mortalities were observed in the pond 3 months after a 30-day 

flow-through exposure of Corbicula to 3.6 to 30 jug/1 of antimycin. A single treatment (2 /ig/1) in 

an earthen pond did not result in significant mortalities of Corbicula during 22 weeks. Magnonaias was more sensitive than Corbicula to antimycin, but both survived the maximum permissible use-pattern concentrations in flow-through tests.

Asiatic clams (several species of the genus Corbicula) introduced into U.S. waters have spread to many 

important river systems (Sinclair and Isom 1963; 

Keup et al. 1963; Diaz 1974). Asiatic clams are becoming so numerous that they cause problems in water intakes to industrial plants, in the pipes and plumbing of 

municipal water supplies, in sand and gravel operations, and as competitors with native species of clams 

for habitat and food.

The widespread distribution of Asiatic clams suggests hardiness and an ability to tolerate adverse environmental conditions. Sinclair and Isom (1963) indicated that these clams can survive fluctuating environmental conditions, and Chandler and Marking 

(1975) reported that they are more resistant than native clams to the lampricide, 3-trifluoromethyl-4-nitrophenol (TFM). Burress et al. (1976) found Asiatic 

clams useful in laboratory toxicity tests because they 

are hardy (Corbicula leana survived for 18 months in

outdoor plastic pools with no water exchange) and can 

be easily collected, transported, handled, and exposed 

to chemicals.

Magnonaias boykiniana, the other clam used in the 

present studies, is native to many American river systems, including those in Georgia.

Pond studies with the fish toxicant, antimycin, 

showed that applications of 5 /*g/l severely reduced 

plankton populations, whereas benthic invertebrates 

were not severely reduced (Callaham 1968; Callaham 

and Huish 1968). Field applications of antimycin in 

Wisconsin were reported to have resulted in delayed 

mortality of several species of mollusks in the East 

Branch, Rock River (Bratley and Mathiak 1972) and in 

the Ashippum River (Flowers et al. 1975). A comprehensive summary of data on the effects of antimycin 

on nontarget organisms prepared by Schnick (1974) indicated that the 96-h LC50 for antimycin against 

Asiatic clams was 50
Antimycin is generally applied at concentrations 

ranging from 1 to 10 jtg/1 in single applications for the 

control of undesired fish populations in ponds and 

lakes, or fpr up to 12 h in streams. The present study 

was designed to determine the acute toxicity and 

latent mortality resulting from short- and long-term 

exposures of the Asiatic clam, Corbicula leana, and a 

native clam, Magnonaias boykiniana, to antimycin. 

Concentrations and exposures investigated equaled or 

exceeded those currently used for fish control.

Materials and Methods

The clams used in this study were collected from 

shoals of the Flint River east of Woodbury (Upson 

County), Georgia. They were transported to the laboratory and kept in limed flowing water until exposed 

to the toxicant. Deformed organisms or any that appeared weakened by handling were discarded. Mature 

specimens of uniform size were selected for the tests.

Clams were exposed to antimycin (Fintrol Concentrate formulation) in limed spring water (total hardness 18-22 mg/1 as CaCO3 and pH 6.8) in four types of 

tests.

(1) Standardized static laboratory tests were conducted in three series of 19-liter glass jars containing 

15 liters of water. The substrate was mud in one series 

and sand in a second; one series of jars contained no 

substrate. Ten Asiatic clams were exposed to 5, 10, or 

20 jtg/1 of antimycin for 1 or 30 days. Antimycin was 

prepared in concentrated stock solutions and portions 

were added to the test water to obtain desired concentrations. Tests were conducted according to recommendations of the Committee on Methods for Toxicity 

Tests with Aquatic Organisms (1975). Surviving clams 

were placed in cages in untreated, previously filled 

earthen ponds for observation over a 112-day period.

(2) An earthen 0.04-ha pond was treated with 2 jtg/1 

of antimycin by dispensing a solution of the toxicant 

into the propeller wash of an outboard motor. Mortality was recorded among the 300 stocked Asiatic 

clams and 85 native clams (weight range, 200-500 g) 

over a 22-week posttreatment observation period. The 

pond water had the following characteristics: total 

hardness as CaCO3, 2.0-14.0 mg/1; pH 5.6-7.4; temperature 6.0-19.0C; and dissolved oxygen 

8.7-15.0 mg/1.

(3) Standardized flow-through toxicity tests were 

conducted in an apparatus similar to that described by 

Mount and Brungs (1967). Asiatic clams were exposed 

to 3.6 to 30 ugll of antimycin in 50-liter glass aquaria 

for 30 days. Thirty clams were exposed to each concentration. The stock solution of antimycin was metered 

into a mixing box, and dilutions of the mixture yielded

selected concentrations. Mortalities were recorded 

daily during exposure.

(4) Flow-through tests were conducted in fiberglass 

tanks in which 300 Asiatic and 75 or 85 native clams 

were exposed to 0.2 and 5 ugll of antimycin for 12 h or 

to 50 figH for 24 h. After exposure, the clams were observed in untreated pond water for 22 weeks. Antimycin solutions were mixed in epoxy-coated steel 

tanks containing 3,623 liters of limed spring water and 

pumped through the tanks at a rate of 4.4 liters/min.

Small amounts of boiled trout chow and cereal 

leaves (Daphnia food) were offered weekly to the 

Asiatic clams in long-term static and flow-through exposures, and excess food was routinely siphoned off.

Survivors of each test were acclimated to holding 

pond temperatures and placed in cages submersed in 

the ponds. Each cage contained a 2-cm layer of soil 

substrate obtained from the holding pond. Cages were 

placed so that water continuously covered the substrate in the cage to a depth of 41 to 46 cm.

The clams were moved to the holding ponds and 

examined semimonthly after exposure. Clams that 

were unable to retract the foot or adduct the valves 

were considered dead. The period between mortality 

determinations was usually long enough so that only a 

casual observation was required because the soft 

tissues of dead specimens protruded from the valves. 

All mortality tabulations are cumulative.

Results

Corbicula survived 1-day exposures to 5, 10, and 

20 ugll of antimycin in 19-liter glass jars containing no 

substrate or substrates of sand or mud (Table 1). 

During the 29-day posttreatment holding period in 

jars containing untreated water, no clams died that 

had been exposed with no substrate, or with mud substrate; however, 70% of the clams died that had been 

exposed to 20 ugll of antimycin in jars with sand substrate. During the later 112-day period in the holding 

pond, a few more died that had been exposed in jars 

with a sand substrate.

Clams exposed for 30 days to 5,10, or 20 jig/1 of antimycin were affected especially during the posttreatment observation period. By the end of the 112-day 

holding period in earthen ponds, all clams had died, 

except for 4 of 10 exposed to 5 ugll with a sand substrate; thus exposure time was a critical factor.

Antimycin was generally not toxic in 1-day exposures, but latent mortality developed in the 30-day exposures. The 1-day exposures typify field use patterns, and 30-day exposures greatly exceed those used 

in fishery management.
Table 1. Cumulative percentage mortality of Corbicula leana after static exposures of 1 day or 30 days to selected concentrations ofantimycin. Each jar contained 10 clams.

Substrate and 

concentration of 

antimycin (/tg/1)

No substrate

0

0

5

5

10

10

20

20

Sand

0

0

5

5

10

10

20

20

Mud

0

0

5

5

10

10

20

20

Exposure 

period 

(days)

1

30

1

30

1

30

1

30

1

30

1

30

1

30

1

30

1

30

1

30

1

30

1

30

Daysinjarsa

30

0

0

0

10

0

50

0

50

0

0

0

0

10

10

70

20

0

10

0

20

0

50

0

40

Days in untreated holding 

pond after exposure

28

0

0

0

30

0

80

0

60

0

0

0

20

10

20

70

20

0

10

0

20

0

50

0

40

70

0

10

0

80

0

100

0

100

0

0

10

50

20

90

70

90

0

10

0

100

0

70

0

100

112

0

10

0

100

0

100

0

100

10

0

10

60

20

100

70

100

0

10

0

100

0

100

0

100

aClams exposed for 1 day were placed in 15 liters of untreated water in 19-liter jars for 29 days after exposure, and then placed in 

a holding pond with those exposed in jars for 30 days.

In the static pond application, in which Corbicula 

and Magnonaias were exposed to 2 /tg/1 of antimycin 

and the toxicant was allowed to dissipate and detoxify 

with time, 98% of the Corbicula and nearly 65% of the 

Magnonaias survived for 22 weeks after the toxicant 

application (Table 2).

Table 2. Cumulative percentage mortality after 2 to 

22 weeks among 300 Corbicula leana and 85 Magnonaias boykiniana, in a pond treated with 2 ng/l of 

antimycin.

Weeks

Species

Corbicula

Magnonaias

0

0

0

8

1.7

22.4

16

1.7

32.9

22

2.0

35.3

Corbicula survived 30 days of exposure to antimycin 

concentrations of 3.6 to 30 ng/\ in a standardized flowthrough system (Table 3). Exceptions were a single 

mortality at 3.6 ngft and two at 15.1 jig/1. These mortalities probably resulted from stresses other than the 

antimycin, since all clams survived at higher concentrations. Few clams died during the first 86 days after 

they were transferred to the holding pond, but latent 

mortality became significant thereafter. After 

156 days, mortality ranged from 27 to 77% (Table 3). 

The die-off seemed to stabilize toward the end of the 

observation period; the increase in mortality from 

128 to 156 days was minor. Again, the 30 days of continuous exposure that led to latent mortality far exceeded exposure time in field applications.

Survival of Corbicula exposed to 2 and 5 /tg/1 of antimycin for 12 h was high during exposure and during
Table 3. Mortality among 30 Corbicula leana during exposure for 30 days to various concentrations ofantimycin in 

a flow-through system, and cumulative percentage mortality in a holding pond 44 to 156 days posttreatment.

Concentration

0«/1)

0.0

3.6

4.4

5.9

8.3

12.4

15.1

20.7

24.1

30.0

Percentage 

mortality 

during exposure

0.0

3.3

0.0

0.0

0.0

0.0

6.6

0.0

0.0

0.0

Days after end of exposure period

44

0.0

3.3

0.0

0.0

0.0

0.0

6.6

0.0

0.0

3.3

86

0.0

10.0

3.3

0.0

3.3

3.3

16.0

3.3

3.3

6.6

128

0

47

27

23

50

50

63

63

67

77

156

0

53

27

30

50

57

67

73

70

77

the later 22-week observation period in an earthen 

pond; mortality ranged from only 0.7 to 2.7% for exposed clams and was 2.3% for unexposed clams 

(Table 4). The mortality of Corbicula exposed to 50 /ig/1 

of antimycin for 24 h was slightly higher 4% at 

2 weeks and 8.7% after 22 weeks.

Magnonaias was more sensitive than Corbicula to 

antimycin in the 12-h exposures to 2 and 5 /ig/1 and 

the 24-h exposure to 50 /ig/1 in the flow-through tank 

system. Mortality was low among Magnonaias exposed for 12 h during the first 2 weeks of observation, 

but increased to 20 to 53% after 22 weeks (Table 4). 

During the 22-week period, however, 20% of the unexposed clams also died. The concentrations of 5 /ig/1 of 

antimycin for 12 h and 50 /ig/1 for 24 h killed more than 

half of the clams during the 22-week observation 

period. Despite these losses it seems clear that the 

population would not have been eliminated, even by 

24-h exposures to 50 /ig/1.

Discussion

Exposure times longer than 12 h and concentrations 

of antimycin higher than 10 /ig/1 are rare in stream 

treatments with antimycin. The registered label prescribes concentrations of 1 to 10 /*g/l, depending on 

physical and chemical conditions of the water. Our exposures of 30 days were excessive. In streams the toxicant is added to flowing water, where concentrations 

build up to a peak of short duration and then decrease 

as a result of dilution and decomposition. Stream organisms are thus exposed to a slug of the toxicant 

passing downstream for an exposure time that varies 

with the species of target fish, and with environmental 

conditions (Gilderhus 1972).

Table 4. Cumulative percentage mortality among 300 

Corbicula leana and 75 or 85 Magnonaias boykiniana exposed to antimycin for 12 or 24 h in flowthrough tanks and moved to an earthen pond for observation for 22 weeks.

Species and 

concentration

Corbicula

0

2

2

5

50

Magnonaias 

0

2

2

5

50

Exposure 

(h)

12

12

12

12

24

12

12

12

12

24

2

0.3

0.0

0.6

4.0

1.3

0.0

3.5

2.4

Weeks

8

0.3

0.7

2.7

1.7

6.0

13.3

9.3

12.9

35.3

30.6

16

1.7

0.7

2.7

1.7

8.7

18.7

17.3

23.5

50.6

48.2

22

2.3

0.7

2.7

2.0

8.7

20.0

20.0

34.1

52.9

50.6

References

Bratley, D. A., and H. A. Mathiak. 1972. Mussels. Pages 

94-97 in U.S. Department of the Interior Draft Environmental Statements: Fishery rehabilitation of the Rock 

River, Dane, Dodge, Columbia, Fond du Lac, Green Lake, 

Jefferson, Rock, Washington, Walworth, and Waukesha 

Counties, Wisconsin. U.S. Fish Wildl. Serv., Washington, 

D.C. DES [Draft Environ. Statement] 72-77.

Burress, R. M., J. H. Chandler, and L. L. Marking. 1976. Use 

of the Asiatic clam, Corbicula leana Prime, in toxicity 

tests. Prog. Fish-Cult. 38(1):10.
Callaham, M. A. 1968. Antimycin as a fisheries tool in the 

Southeast. Ph.D. Thesis. University of Georgia, Athens. 

76pp.

Callaham, M. A., and M. T. Huish. 1968. An evaluation of 

antimycin as a selective bluegill toxicant under varying 

conditions of pH. Proc. Annu. Conf. Southeast. Assoc. 

Game Fish Comm. 21(1967):476-481.

Chandler, J. H., and L. L. Marking. 1975. Toxicity of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to selected 

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

Serv., Invest. Fish Control 62. 7 pp.

Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, 

macroinvertebrates, and amphibians. Ecol. Res. Series. 

EPA [Environ. Prot. Agency]-660/3-75-009. 61 pp.

Diaz, R. J. 1974. Asiatic clam, Corbicula manilensis (Philippi), 

in the tidal James River, Virginia. Chesapeake Sci. 

15(2):118-120.

Flowers, R. W., R. M. Johnson, and W. L. Hilsenhoff. 1975. 

Effect of antimycin on instream mussel populations. Wis. 

Dep. Nat. Resour. Res. Rep. 84.11 pp.

Gilderhus, P. A. 1972. Exposure times necessary for antimycin and rotenone to eliminate certain freshwater fish. J. 

Fish Res. Board Can. 29(2):119-202.

Keup, L., W. B. Horning, and W. M. Ingram. 1963. Extension 

of range of Asiatic clam to Cincinnati reach of the Ohio 

River. Nautilus 77(1):18-21.

Mount, D. L, and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Res. 1:21-29.

Schnick, R. A. 1974. A review of the literature on the use of 

antimycin in fisheries. FWS [Fish Wildl. Serv.] LR [Lit. 

Rev.] 74-01. NTIS [Natl. Tech. Info. Serv.] No. PB-235, 

440/AS. 85 pp.

Sinclair, R. M., and B. G. Isom. 1963. Further studies on the 

introduced Asiatic clam (Corbicula) in Tennessee. Tennessee Stream Pollution Control Board, Nashville. 78 pp.

Chronic and Simulated Use-Pattern Exposures 

of Brook Trout (Salvelinus fontinalis) to 

3-Trifluoromethyl-4-nitrophenol (TFM)

by

W. P. Dwyer 1 , F. L. Mayer, J. L. Alien, and D. R. Buckler

U.S. Fish and Wildlife Service

Columbia National Fisheries Research Laboratory

Columbia, Missouri 65201

and

(J.L.A.) Fish Control Laboratory 

La Crosse, Wisconsin 54601

Abstract

Effects of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) on brook trout (Salvelinus fontinalis) were compared under conditions of continuous (chronic) exposure, and under conditions 

simulating those used in the application of TFM in tributary streams of the Great Lakes for control of the sea lamprey (Petromyzon marinus). Chronic exposure of adult brock trout to concentrations of 4.0 mg/1 or higher caused deleterious effects on growth, spawning, survival during 

spawning, and eye condition. Hatchability and viability of the eggs were reduced. Growth and 

survival were reduced in the fry at TFM concentrations of 1.6 mg/1 or higher. No deleterious effects were noted at lower concentrations. The only effect observed in fish after simulated usepattern exposure to TFM was a decrease in survival of adults tested at 15 C.

The lampricide 3-trifluoromethyl-4-nitrophenol 

(TFM) was registered in 1964 for control of larval sea 

lampreys (Petromyzon marinus) in selected tributaries 

of the Great Lakes. The U.S. Environmental Protection Agency (EPA) is now reviewing the registration of 

TFM. A substantial amount of research has been conducted on TFM toxicity and selectivity to sea lamprey 

larvae, as indicated by the literature reviewed by 

Schnick (1972). Later research on toxicity included 

work with algae (Maki et al. 1975), midge larvae 

(Kawatski et al. 1975), mayfly nymphs (Fremling 

1975), aquatic invertebrates and frog larvae (Chandler 

and Marking 1975), and early life stages of rainbow 

trout (Olson and Marking 1973); other studies were on 

toxicity and residue dynamics in invertebrates 

(Sanders and Walsh 1975), and included tests on nontarget fish both static (Marking and Olson 1975) 

and flow-through (Marking et al. 1975).

Present address: Fish Cultural Development Center, Bozeman, Montana 59715.

The accumulation and elimination of TFM residues 

by fish exposed to TFM after a lamprey control treatment of the East Au Gres River, Michigan, were reported by Gilderhus et al. (1975). Biotransformation 

and elimination of TFM by fish were elucidated by 

Lech and Costrini (1972). Hunn and Alien (1974, 1975) 

reported on the factors affecting the uptake and elimination of the lampricide, and the renal excretion of the 

compound in fish.

The objectives of the present research were to determine the effects of TFM on brook trout (Salvelinus 

fontinalis) under continuous (chronic) exposure and 

under exposures simulating a use pattern to which fish 

would be exposed during a stream treatment. Brook 

trout are an important and indigenous sport fish in 

many of the streams treated with TFM. Factors 

measured were mortality, growth, and reproduction. 

Samples of fish were analyzed to determine accumulation and elimination of TFM after continuous and simulated use-pattern exposures.
Materials and Methods

Chronic Study

Yearling brook trout obtained from the Manchester 

(Iowa) National Fish Hatchery were held in raceways 

at 16 C and fed a maintenance diet (Brauhn and 

Schoettger 1975) until the tests were started. On 

14 June 1973, 12 fish were placed in each of 12 stainless steel tanks, measuring 137 cm long, 36 cm wide, 

and 51 cm high, in which water depth was 30 cm. Well 

water was delivered to the tanks at a rate of 

800 ml/min per tank through a proportional diluter 

system modeled after Mount and Brungs (1967). Each 

tank was aerated with filtered air to maintain oxygen 

concentrations above 70% saturation. Adult fish were 

fed a modification (Mehrle et al. 1977) of the Oregon 

Test Diet (National Academy of Sciences 1973) ad libitum throughout the study. Fry were fed the commercial trout starter "Ewos."

A diluter system with the modification of McAllister 

et al. (1972) and a dilution factor of 0.5 between concentrations was used to continuously deliver five concentrations of TFM and a control for the chronic test. 

The TFM used was a 35.7% field grade formulation 

manufactured by the American Hoechst Chemical 

Company; distilled water was the carrier solvent. The 

measured TFM concentrations (total formulation) 

averaged 0.94, 1.6, 4.0, 8.8, and 16 mg/1 (or 0.34, 0.57, 

1.4, 3.1, and 5.7 mg/1 active ingredient). Flow-splitting 

chambers designed by Benoit and Puglisi (1973) were 

used to thoroughly mix and divide each lampricide 

concentration for delivery to duplicate exposure tanks. 

The water temperature in the tanks was controlled by 

refrigeration units suspended in a circulating water 

bath. Artificial daylight was provided by the method 

of Drummond and Dawson (1970), and the water temperature regime and photoperiod were those recommended for brook trout tests (EPA 1972).

The study was conducted according to the recommended procedures for partial chronic tests with brook 

trout by EPA (1972). The fish were measured (total 

length) after 60 and 120 days of exposure. After the 

final growth determinations, the fish were thinned to 

five to eight per tank and two spawning substrates 

were placed in each tank. The excess fish above three 

females and two males were used for artificial reproduction (stripping of eggs and sperm). Spawning 

began 28 November 1974 and ended 16 January 1975. 

Two samples of 100 eggs from each spawning, both natural and artificial, were placed in incubator cups to determine hatchability and viability. Viability was determined 10 days after spawning by placing the eggs in 

10% acetic acid for several minutes until the neural 

keel became visible, indicating that the embryo was developing. Upon hatching, 25 fry were randomly selected from each incubation cup, their length was determined by the photographic method of McKim and 

Benoit (1971), and they were transferred to growth 

chambers 14 cm deep, 38 cm long, and 15 cm wide, in 

which water was 10 cm deep. The fry were also 

measured photographically at 30 and 60 days, and 

measured directly at 90 days. Mortality of fry was recorded daily.

Use-Pattern Study

The use-pattern study was initiated a year after the 

chronic study, with 2-year-old fish from the same lot 

used in the chronic study. The procedures were essentially the same, except that the exposure of fish to 

TFM was designed to simulate stream treatment for 

lamprey control. In such treatments, TFM is metered 

into streams at concentrations of 2 to 20 mg/1 (total 

formulation) for 10 to 12 h, depending on water quality 

and on-site bioassay. A stream is normally treated 

once during the summer or fall. In the simulated usepattern study, eight fish were placed into each of six 

tanks. TFM was metered into two tanks for 12 h at 

18 mg/1 on 30 July 1974, and into two other tanks at 

16 mg/1 on 1 October 1974; the remaining two tanks 

served as controls. The diluter system used to expose 

the fish was that of Brungs and Mount (1967). Since 

egg viability and hatchability did not differ between 

eggs naturally and artificially spawned in the chronic 

study, spawning was expedited in the use-pattern 

study by hand stripping sexually mature brook trout. 

Egg viability and hatchability, and fry growth were 

measured as in the chronic study.

Residue Analysis

Residues of TFM were determined on both fillet and 

offal of four adult fish from each concentration after 

137 days of continuous exposure. Six additional fish 

from each of the 1.6- and 4.0-mg/l exposures were 

transferred to uncontaminated water at 9 C to determine the elimination rate of TFM; two fish were 

sampled from each exposure after 3, 7, and 14 days in 

fresh water. A sample of eggs from each of three 

spawns within each TFM concentration was also 

analyzed. Three fish from the controls and the last exposure of the use-pattern study were sampled 45 days 

after that exposure. Fish and eggs were prepared for 

residue analysis by the method of Benville and Tindle 

(1970) and extracted by the column chromatography 

technique of Hesselberg and Johnson (1972). The extracts were analyzed by gas chromatography (Alien 

and Sills 1974), in which the minimum detection limit 

was 1 ng/g. (However, TFM residues below 10 ng/g
were not quantifiable.) Concentrations of TFM in 

water were periodically determined by the colorimetric 

method of Olson and Marking (1973).

Experimental Design and Analysis

The design of both tests was a randomized block. 

Growth data (length gained) were analyzed by analysis 

of variance (Snedecor 1965). A multiple means comparison test (least significant difference) was used to 

compare treatments. The effects of TFM on fish mortality and egg hatchability and viability were determined by conducting an analysis of variance on the 

arcsin transformation for proportions (angle = arcsin 

V percentage ), followed by least significant difference 

tests.

Results

No differences in adult growth were detected after 

60 days of chronic exposure to TFM. However, growth 

was significantly reduced (P < 0.05) after 120 days in 

fish exposed to 8.8 and 16 mg/1 TFM (Table 1). Growth 

of adult fish under use-pattern conditions was not affected. Chronic exposure to high concentrations of 

TFM may have caused blindness in some adult fish. 

The eye was covered with an opaque layer and, in some 

fish, was filled with blood. After 120 days exposure to 

16 mg/1 TFM, 10% of the fish were affected in at least 

one eye; by 180 days, 42% of the fish were affected. Although none of the other fish exposed to lower concentrations showed similar effects at 120 days, a few fish 

exposed to 8.8 mg/1 TFM developed the eye condition 

at 180 days.

As total spawning activity increased with time, so 

did the mortality of the adults exposed to 16 mg/1 

TFM and (to a lesser extent) of those exposed to 

8.8 mg/1 (Table 2). Apparently the fish died from 

spawning stress in addition to the TFM exposure. At 

death, the fish were covered with copious amounts of 

mucus. Under use-pattern conditions, 19% of the adult 

fish died during the early exposure, when the water 

temperature was 15 C. No mortalities occurred at 9 C, 

just before the fish spawned, in the late exposure.

In the chronic-exposure study, no spawns were obtained from the fish exposed to 16 mg/1 TFM, and only 

one natural spawn occurred at 8.8 mg/1 (Table 3). However, these eggs were not viable. Inasmuch as egg viability and hatchability of natural and artificial 

spawns were not statistically different, the viability 

and hatchability data were pooled (Table 4). The viability of eggs in the 4.0 mg/1 TFM concentration was 

statistically less (P < 0.05) than that of eggs in the 

lower concentrations and controls. Hatchability of 

eggs at this concentration was also lower, although

Table 1. Mean total lengths (mm; standard deviations 

in parentheses) of adult brook trout before, and 

120 days after chronic exposure of yearlings and 

100 days after use-pattern exposure of 2-year-olds, 

to different concentrations of TFM.

Type of exposure, and 

concentration of TFM

(mg/1)-

Length (mm) of fish:

On day 120 (chronic) 

On day 0 or 100 (use-pattern)

Chronic

0.0

0.94

1.6

4.0

8.8

16.0

Use-pattern

0.0

18.0

16.0

213(14)

215(12)

218(15)

213 (22)

220(13)

220(11)

300 (27)

306 (28)

312(36)

267 (16)

265(17)

268(17)

265(10)

264(15)b

253 (18)b

316 (28)

324 (30)

320 (30)

a Total formulation.

b Length gained significantly different (P < 0.05) from that 

of controls.

Table 2. Mortality of brook trout after chronic and usepattern exposures to TFM.

Type of exposure, and 

concentration of TFM

(mg/l)a

Mortality (%)

Adults Fry

Chronic

0.0

0.94

1.6

4.0

8.8

16.0

Use-pattern

0.0

18.0

16.0

0

0

0

0

34b

68b

0

19b

0

27

58

84b

100b

100b

100b

14

6

9

a Total formulation.

b Significantly different from controls (P < 0.05).

not significantly so. In addition, the eggs exposed to 

4.0 mg/1 TFM were not consistent in size, shape, and 

neural keel development, when compared with those 

exposed to lower concentrations, and the control. The 

TFM had no effect on viability or hatchability of eggs 

from adults treated in the use-pattern exposures.
Table 3. Production of spawn by brook trout after chronic and use-pattern exposures to TFM.

Type of exposure, and

concentration of TFM

(mg/1)"

Chronic

0.0

0.94

1.6

4.0

8.8

16.0

Use-patternb

0.0

18.0

16.0

Number

7

6

8

8

1

0

-

Natural spawns

Eggs per spawn

(number)

309

294

215

236

143

Artificial

spawns

(number)

2

4

3

1

0

0

5

4

5

"Total formulation.

bEggs per spawn were not considered because the fish were stripped to obtain eggs and sperm.

Table 4. Viability and hatchability of brook trout eggs 

after chronic and use-pattern exposures of TFM.

Type of exposure, and

concentration of TFM Viability Hatch

(mgA)8 (%) (%)

Chronic

0.0

0.94

1.6

4.0

8.8

16.0

Use-pattern

0.0

18.0

16.0

82

87

88

50b

Ob

 C

92

97

97

67

67

74

43

82

88

85

a Total formulation.

bSignificantly different from controls (P < 0.05).

c No spawns produced.

For the first 60 days of chronic exposure, no statistical difference existed in the growth of fry continuously exposed to TFM; however, at 90 days growth 

was significantly reduced (P < 0.05) in fry exposed to 

1.6 mg/1 TFM (Table 5). The length gained in fry exposed for 90 days to 0, 0.94, and 1.6 mg/1 TFM was 

35.2, 32.7, and 25.8 mm, respectively. All fry exposed 

to 4.0 mg/1 TFM were dead within 30 days (Table 2). 

No significant differences in mortality of fry exposed 

to the two lower concentrations and controls were observed at 30 days of exposure. At 60 and 90 days, however, the mortality of fry exposed to 1.6 mgA TFM was 

significantly higher (P < 0.05) than that of the controls. Growth and survival of fry were not affected in 

the use-pattern study.

Table 5. Mean total lengths (mm; standard deviations 

in parentheses) of brook trout fry 90 days after 

chronic exposure to different concentrations of 

TFM or fry from parents exposed to use-pattern 

concentrations of TFM.

Type of exposure, and 

concentration of TFM

(mg/1)*

Length (mm) of fish:

On day 0 On day 90

Chronic

0.0

0.94

1.6

4.0

8.8

16.0

Use-pattern

0.0

18.0

16.0

14.8 (0.6)

14.3(1.0)

15.2(0.9)

_ c

_d

_ e

16.2(0.9)

16.5(1.1)

16.7(0.7)

50(2)

47(4)

41 (6)b

49(5)

51(4)

49(3)

a Total formulation.

bSignificantly different from controls (P < 0.05) based on

length gained. 

c Fry died within 30 days. 

dNo eggs hatched. 

eNo spawns produced.
Table 6. Mean concentrations of TFM (ng/g; standard error in parentheses) in fillets and offal of adult brook trout 

and their eggs, after continuous exposure to different concentrations of TFM for 137 days.

Concentration of 

TFM (mg/l)a

0.0

0.94

1.6

4.0

8.8

16.0

Fillets 

<n = 4)

<0.01

0.01 (0.003)

0.06 (0.01)

0.09 (0.01)

0.08 (0.04)

0.11(0.03)

Offal 

(n = 4)

<0.01

0.06 (0.02)

0.43(0.10)

0.19(0.03)

0.18 (0.05)

0.38(0.11)

Eggs 

(n = 3)

<0.01

0.18 (0.02)

0.32 (0.03)

0.74 (0.17)

1.9b

2.8 (0.49)«=

"Total formulation.

bn = l.

c Eggs for samples were removed from females since there were no natural spawns in this exposure.

Residues of TFM in the eggs of brook trout in the 

chronic-exposure study averaged 0.18 uglg in fish exposed to 0.94 mg/1 TFM and 2.8 uglg in fish exposed to 

16 mg/1 (Table 6). The fillet and remaining offal from 

adult brook trout were analyzed separately. TFM in 

the fillets ranged from 0.01 uglg for fish exposed to 

0.94 mg/1 TFM to 0.11 uglg for fish exposed to 16 mg/1, 

indicating an accumulation factor of 0.02 to 0.1, based 

on the active ingredient. Residues of TFM in the offal 

from adult brook trout varied considerably, but the indicated accumulation factor was 0.8 or less for each of 

the exposure concentrations. No residues were found 

under use-pattern conditions. Results from the residue 

elimination phase of the experiment indicate that the 

loss of TFM was rapid (Table 7).

Discussion

Chronic exposure of adult brook trout to TFM concentrations of 4.0 mg/1 or less caused no significant differences in growth, but concentrations of 8.8 and 

16 mg/1 resulted in reduced growth and number of 

spawns, and adversely affected the eyes. Viability and 

hatchability of eggs, consistency of egg size and shape, 

and embryo development were affected by exposure to 

4.0 mg/1 of the lampricide. Some mortality occurred in

the 8.8 and 16 mg/1 concentrations, apparently in part 

as a result of the added stress of spawning. This same 

response was observed in adult brook trout exposed to 

toxaphene (Mayer et al. 1975). Effects on growth and 

mortality were noted in fry exposed to 1.6 mg/1 or 

more of TFM but no significant effect was noted between fry exposed to 0.94 mg/1 and controls. No significant differences were found between controls and 

fish exposed to use-pattern treatments, except for the 

increased mortality of adults.

The use pattern of TFM as a lamprey larvicide precludes long-term and continuous exposure of fish to 

concentrations that might cause the effects shown in 

this study. Gilderhus et al. (1975) showed that the concentration of TFM in water dropped rapidly after termination of the treatment of the East Au Ores River, 

Michigan. Coburn and Chau (1976) reported 40 ugll of 

TFM in a low-flow area of a creek 3 weeks after the 

treatment for lamprey control. Our studies show no 

effect on brook trout continuously exposed to about 

9.5 times this amount (0.94 mg/1 or 0.34 mg/1 active ingredient).

The residues in fillets and offal of adults after 

chronic exposure to TFM showed a low accumulation 

factor and TFM was rapidly eliminated when the fish 

were transferred to uncontaminated water. The residue elimination followed a pattern similar to that reTable 7. Elimination of whole-body TFM residues (ng/g) in adult brook trout transferred to uncontaminated water

after 137 days of continuous exposure to 1.6 or 4.0 mg/l TFM.

Concentration of 

TFM (mg/l)a

1.6 

4.0

3

0.06 

0.04

Days in uncontaminated water

7

<0.01 

0.02

14

<0.01 

<0.01

"Total formulation.
ported by Sills and Alien (1975). No residues were 

found in adult fish at the time of spawning in the usepattern exposure.

References

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Assoc. Off. Anal. Chem. 57:387-388.

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

Serv., Invest. Fish Control 62. 7 pp.

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method for simulating diel patterns of lighting in the laboratory. Trans. Am. Fish. Soc. 99:434-435.

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(Hexagenia sp.). U.S. Fish Wildl. Serv., Invest. Fish Control 58. 8 pp.

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3-trifluoromethyl-4-nitrophenol (TFM) in a stream ecosystem after treatment for control of sea lamprey. U.S. 

Fish Wildl. Serv., Invest. Fish Control 66. 5 pp.

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the gills of fishes. Annu. Rev. Pharmacol. 14:47-55.

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6:15-18.

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

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metabolism of 3-trifluoromethyl-4-nitrophenol (TFM) in 

rainbow trout. Comp. Gen. Pharmacol. 3:160-166.

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

10 species of algae. U.S. Fish Wildl. Serv., Invest. Fish 

Control 56.17pp.

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of the lampricide trifluoromethyl-4-nitrophenol (TFM) to 

nontarget fish in flow-through tests. U.S. Fish Wildl. 

Serv., Invest. Fish Control 61. 9 pp.

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fish in static tests. U.S. Fish Wildl. Serv., Invest. Fish 

Control 60. 27 pp.

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brook trout. U.S. Environ. Prot. Agency, (Duluth, Minn.) 

Ecol. Res. Ser. EPA-600/3-75-013. 51 pp.

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

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of domestic animals. No. 11. Nutrient requirements of 

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(lampricide) to six early life stages of rainbow trout (Salmo 

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Fish Wildl. Serv., Invest. Fish Control 44. 31 pp.

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

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

Invest. Fish Control 65. 5 pp.

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Hydrolysis and Photolysis of the Lampricide 

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

by

D. P. Schultz and P. D. Harman

U.S. Fish and Wildlife Service

Southeastern Fish Control Laboratory

Warm Springs, Georgia 31830

Abstract

The hydrolysis and photolysis of the lampricide Bayer 73 (aminoethanol salt of 2',5-dichloro-4'- 

nitrosalicylanilide) was studied by using 14C-Bayer 2353 (the non-salt form of Bayer 73). No 

hydrolysis of 14C-Bayer 2353 occurred in pond water or in distilled water buffered at pH 5.0, 6.9, 

or 8.7 after 56 days. During exposure to long-wave UV light, MC-Bayer 2353 degraded rapidly on 

silica gel thin layer chromatographic plates and on glass slides. After exposures of 24 and 168 h, 

less than 50 and 5%, respectively, of the remaining radioactivity was parent compound. After exposure of an aqueous solution of 14C-Bayer 2353 to long-wave UV light for 14 days, only 5% of the 

remaining radioactivity was parent compound.

Bayer 73, the aminoethanol salt of 2',5-dichloro-4'- 

nitrosalicylanilide (Bayer 2353), is not only toxic to 

larvae of the sea lamprey (Petromyzon marinus) but 

also synergizes the activity of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) and increases its effectiveness (Howell et al. 1964).

The requirement that all piscicides used in the United States must be registered with the Environmental 

Protection Agency includes the development of data 

concerning the fate of the chemical in the environment 

and potential rates of hydrolysis and photolysis. In 

this study, we investigated the hydrolysis of 14C-Bayer 

2353 in water buffered at various pH's, and studied 

the photolysis of 14C-Bayer 2353 on thin layer chromatographic plates, on glass slides, and in aqueous 

solutions.

General Materials and Methods

In all tests, materials that had been exposed to UV 

light were spotted on thin layer chromatographic 

(TLC) plates (silica gel, F-254, 250 /on; Brinkman Instruments, Westbury, N.Y.). The plates were developed in a solution of chloroform:methanol:ammonium hydroxide (50:20:2.5, v/v/v), dried, and placed 

on X-ray films. After 3 to 4 weeks of exposure, the Xray films were developed, the R, (distance compound 

moved on TLC plate/distance solvent moved on TLC 

plate) of each spot was determined, and corresponding 

radioactive spots on the TLC plates were scraped into

scintillation vials for radioactive determination. Nonradioactive scrapings of TLC plates were used to determine quench and background. The scintillation 

cocktail consisted of Beckman TLA dissolved in 

toluene.

Hydrolysis of Bayer 73

To investigate the hydrolysis of Bayer 73, we used 

three 250-ml beakers containing 100 ml of distilled 

water buffered (buffer tablets, No. 13-640-304-E,K,H, 

Fisher Scientific, Atlanta, Ga.) to a pH of 5.0, 6.9, or 

8.7, and a fourth beaker containing 100 ml of pond 

water (initial pH, 7.8). The pH of the buffered solutions 

did not change during the experiment; that of the pond 

water decreased from pH 7.8 to 7.0. Into each beaker 

we stirred 1 ml of acetone containing 5 fig of 14C-Bayer 

2353 (chlorosalicyclic acid ring UL-14C, specific activity, 10 mCi/mmol, American Radiochemical Corporation, Sanford, Fla.). The acetone was allowed to 

evaporate, and the beakers were then covered with 

aluminum foil and kept in the dark at 20 ± 1 C. 

Samples (100 pi) were taken at 0,1, 4, 7, 14, 26, and 56 

days, and spotted on thin layer chromatographic 

plates for separation and quantification of possible 

degradation products.

No degradation of 14C-Bayer 2353 occurred within 

the 56 days in either the buffered aqueous solutions or 

in the pond water (Table 1).
Table 1. Amounts of l4C-Bayer 2353 and l4C-degradation product from TLCplates spotted with aqueous solutions 

of various pH values, and pond water containing l4C-Bayer 2353 and aged for 0 to 56 days (values expressed as 

percentages of total radioactivity from each plate).

Buffered water

Days

0

1

4

7

14

26

46

56

R/a

0

0.78

0

0.78

0

0.78

0

0.78

0

0.78

0

0.78

0

0.78

0

0.78

pH5.0

2

98

4

96

1

99

1

99

1

99

1

99

1

99

6

95

pH6.9

5

95

6

94

5

95

2

98

4

96

5

95

1

99

2

98

pH8.7

5

95

5

95

3

97

2

98

2

98

3

97

3

97

2

98

Pond 

(pH 7.0)

1

99

1

99

3

97

1

99

7

93

6

94

4

96

4

96

Standard 

( 14C-Bayer 2353)

1

99

1

99

1

99

1

99

1

99

1

99

1

99

1

99

aO = origin; 0.78 = parent compound, 14C-Bayer 2353.

Photolysis of Bayer 73

Photolysis on Silica Gel Plates

A solution of 14C-Bayer 2353 (1.31 fig in 10 jJ of 

hexane) was spotted on 18 silica gel TLC plates, 9 of 

which (the controls) were wrapped in aluminum foil 

and 9 were left uncovered. The plates were exposed at 

a distance of 20.3 cm to a long-wave lamp (Blak-Ray, 

Model X-30, Ultra-Violet Products, Inc., San Gabriel, 

Calif.) with wave lengths ranging from 290 to 405 nm 

and a peak emission at 355 nm. The intensity at the 

surface of the TLC plates, measured with a Blak-Ray 

UV Meter (Model J221, Ultra-Violet Products, Inc., 

San Gabriel, Calif.) was 9,800 ergs/s per cm2 . One control and one exposed plate were removed from under 

the light at 0, 1, 2, 4, 6, 24, 48, 120, and 168 h and prepared for R, determination and radiometric quantifications.

The 14C-Bayer 2353 degraded rapidly on the TLC 

plates exposed to long-wave UV light (Table 2). About 

15% of the parent compound had been degraded after 

4 h of exposure, 60% after 24 h, and 95% after 168 h 

(Fig. 1). Most of the degraded material remained at the 

origin of the TLC plates; e.g., after a 168-h exposure, 

more than 70% of the radioactivity remained at the 

origin. Several other compounds with R, values of 0.25, 

0.34, 0.41, 0.71, 0.74, and 1.0 (R, of the parent compound was 0.60) were found; their percentages increased up to 24 or 48 h of exposure and then declined. 

However, none of these compounds ever exceeded 8% 

of the total radioactivity. The only major compound 

other than that found at the origin, was a composite of 

compounds with R, values ranging from 0.04 to 0.12. 

These spots were distinct, but overlapped, and could 

not be separated. The R/ of one other spot (0.25) corresponded to that of standard 14C-chlorosalicyclic acid. 

This spot never exceeded 3.8% of the total radioactivity. No attempt was made te^oheck for nonradioactive degradation products such as chloronitroaniline 

because the amount potentially present was very 

small.

Photolysis on Glass Slides

In further tests of the photolysis of Bayer 73, we 

streaked 10 jJ (1.31 ng) of uC-Bayer 2353 on 18 glass 

microscope slides. The solvent was evaporated at room 

temperature in a fume hood, and the slides were then 

placed under long-wave UV as described for the TLC 

plates. One group of nine slides was covered with 

aluminum foil to serve as controls. Individual slides 

from each set were removed from UV exposure at 0, 1, 

2, 4, 6, 24, 48, 120, and 168 h. The radioactive material 

was washed from the slides with 1 ml of acetone: 

methanol (50:50, v/v), and the wash concentrated to 

0.5 ml and spotted on TLC plates for development, R/
Table 2. Amounts of l4C-Bayer 2353 and 14C-degradation products from TLC plates spotted with 14C-Bayer 2353 

and exposed to img-wave UV light for up to 168 h (values expressed as percentages of total radioactivity from 

each plate; values in parentheses are from control plates)".

Time 

(h)

0

1

2

4

6

24

48

120

168

0

0.2

1.9

(0.2)

2.7

(0.2)

4.2

(0.2)

8.5

(0.2)

27.7

(0.3)

44.0

(0.3)

66.9

(0.5)

73.7

(0.4)

O.lb

0.0

1.6

2.4

3.4

5.6

10.8

11.4

10.4

11.6

0.25'

0.4

0.8

(0.4)

0.8

(0.4)

0.9

(0.4)

2.4

(0.4)

3.3

(0.4)

3.8

(0.4)

3.2

(0.5)

2.3

(0.4)

0.34

0.0

0.9

1.5

2.4

4.5

8.0

7.5

5.2

3.9

(0.2)

R/

0.41

0.0

0.8

1.3

1.6

2.6

3.9

4.0

2.9

1.6

0.60d

99.0

92.7

(98.7)

89.3

(98.9)

84.5

(98.5)

71.4

(99.1)

39.9

(99.0)

22.7

(98.7)

8.0

(98.4)

4.4

(98.7)

0.71

0.0

0.0

0.0

1.0

1.8

2.2

2.8

1.3

1.1

0.74

0.2

0.9

(0.4)

1.2

(0.3)

1.2

(0.7)

1.6

(0.2)

2.2

(0.2)

2.0

(0.3)

1.0

(0.4)

1.0

(0.3)

Solvent 

front

0.1

0.4

(0.4)

0.9

(0.2)

0.9

(0.2)

1.5

(0.1)

2.0

(0.1)

1.6

(0.2)

1.0

(0.2)

0.3

(0.1)

aPlates exposed to 9,800 ergs/s per cm2 .

bA composite of spots with R/s ranging from 0.04 to 0.12.

cThis R, corresponds to that of 14C-chlorosalicylic acid.

dThis R/ corresponds to that of the parent compound, 14C-Bayer 2353.

determination, and radiometric quantification.

The degradation of 14C-Bayer 2353 on glass slides 

was similar to that on TLC plates (Table 3), except 

that the degradation occurred more rapidly in the first 

24 h. For example, after 4 and 6 h of exposure only 

62.3 and 56.7% of the parent compound remained on 

glass slides, as compared with 84.5 and 71.4% on TLC 

plates. The more rapid rate of degradation could have 

resulted from greater penetration of UV light through 

the chemical on the glass slides, whereas the silica gel 

could have absorbed or adsorbed 14C-Bayer 2353 and 

prevented the light from affecting all the molecules on 

the TLC plates. Material with an R, of 0.1 increased 

during the first 24 h and then declined on glass slides 

whereas the same compound remained nearly constant 

after 24- to 168-h exposures on TLC plates. The change 

in the slope of the degradation curve (Fig. 1) may have 

resulted from incomplete elution of the radioactive 

compounds from the glass slides.

Photolysis in Buffered Solutions

To examine photolysis in buffered solutions, we 

added 1 ml of an acetone solution containing 5 ng of

14C-Bayer 2353 to two 250-ml beakers containing 100 

ml of distilled water buffered to pH 6.9. Depth of the 

solution was 4.3 cm. The beakers were placed in the 

dark until the acetone had evaporated. The control 

beaker was then covered with aluminum foil and the 

other was left uncovered. The beakers were placed in a 

constant temperature water bath maintained at 

20 ± 1 C and exposed to long-wave UV light. The distance from the lamp to the surface of the solution was 

20.3 cm. Samples (100 n\) were taken from each beaker 

at 0, 1, 4, 7, and 14 days and spotted on silica gel TLC 

plates for development, R/ determination, and radiometric quantification.

The 14C-Bayer 2353 in the aqueous solution was also 

degraded by long-wave UV light (Table 4), but the degradation (which was accompanied by a reduction in pH 

from 6.9 to 6.7) was much slower than on the TLC 

plates or glass slides. However, only 49% of the parent 

compound remained after 7 days of exposure and only 

5% after 14 days. Most of the degraded material remained at the origin as on the TLC plates and glass 

slides. Three other compounds with R/ values of 0.13, 

0.34, and 0.41 were noticed on the X-ray films from the 

study of aqueous photolysis. The spot at an R/ of 0.13
Table 3. Amounts of l4C-Bayer 2353 and l4C-Bayer degradation products from TLC plates spotted with eluants 

from glass slides. Glass slides were streaked with l4C-Bayer 2353 and then exposed to long-wave UV light up to 

168 h (values expressed as percentages of total radioactivity from each plate; values in parentheses are from 

control plates)0.

Time 

(h)

0

1

2

4

6

24

48

120

168

0

0.25

1.4 

(0.1)

3.2 

(0.2)

12.8 

(0.2)

13.7 

(0.2)

47.5 

(0.2)

56.7 

(0.2)

65.9 

(0.2)

83.8 

(0.4)

O.lb

0.0

2.5

5.0

9.2

11.2

12.6

10.3

9.5

4.5

0.25C

0.4

1.1 

(0.3)

1.5 

(0.3)

(0.4)

(0.3)

(0.4)

(0.4)

(0.4)

(0.4)

R/

0.34

0.0

2.2

2.6

4.8

6.1

7.3

5.3

6.2

4.8

0.41

0.0

2.6

2.9

5.8

7.2

6.2

5.7

4.8

3.1

0.60.

98.9

86.7 

(98.8)

81.0 

(99.0)

62.3 

(99.2)

56.7 

(99.1)

22.9 

(99.1)

19.7 

(99.1)

12.8 

(99.1)

3.6 

(99.0)

0.71

0.2

1.5

1.4

2.4

2.7

2.2

1.7

0.0

0.0

Solvent 

front

0.3

1.9 

(0.8)

2.4 

(0.5)

2.7 

(0.3)

2.2 

(0.3)

1.3 

(0.3)

0.4 

(0.3)

0.8 

(0.3)

0.1 

(0.2)

aGlass slides exposed to 9,800 ergs/s per cm2 .

bA composite of spots with R/s ranging from 0.04 to 0.12.

This R, corresponds to that of 14C-chlorosalicylic acid.

dThis R/ corresponds to that of the parent compound 14C-Bayer 2353.

appeared only at the 14-day exposure and was a composite of several inseparable spots with R/s ranging 

from 0.09 to 0.16.

Comparison with Other Studies

Strufe and Gonnert (1962) reported that the lowest 

concentration of Bayer 73 lethal to snails increased 

from 0.3 to about 1.8 mg/1 after exposure of the chemical to UV light for 8 h. Meyling et al. (1962), who exposed 1-mg/l solutions of Bayer 73 to direct sunlight 

for up to 36 h, found that the concentrations of Bayer 

73 in soft water dropped from 1 mg/1 at 0 h to 0.9, 0.8, 

0.67, and 0.56 mg/1 at 8, 16, 24, and 36 h, and in hard 

water from 1.0 at 0 h to 0.71, 0.58, and 0.57 mg/1 at 8, 

12, and 16 h. Dawson (1971), who exposed Bayer 73 to 

UV light found that the LC50 to bluegills (Lepomis 

macrochirus) was 0.252 mg/1 after 6 h (as compared 

with a control value of 0.197 mg/1) and 0.348 mg/1 after 

24 h (as compared with a control value of 0.250). He

also reported LC50 's of 0.242 for Bayer 73 exposed to 

sunlight for 6 h, 0.177 for shielded samples, and 0.150 

mg/1 for control samples.

Other workers reported no effects of UV light or sunlight on the toxicity of Bayer 73. Farringer (1972) exposed solutions of Bayer 73 to UV light for up to 96 h 

and found no change in toxicity of the chemical to carp 

(Cyprinus carpio). He believed that the differences between his results and those of Strufe and Gonnert 

(1962) may have been due to the difference in formulations. Farringer used a 99% technical formulation, 

whereas Strufe and Gonnert used a 70% wettable 

powder.

Sturrock (1974), after gathering samples of aquatic 

vegetation which had been sprayed with Bayer 73, reported no diminution in toxicity to snails from samples 

exposed to tropical sunlight for up to 8 weeks. 

Possibly the chemical was "protected" from UV degradation by absorption into, or adsorption onto, the 

plant material.
" 10 -

30 60 90 120 150 168 

HOURS EXPOSURE TO UV LIGHT

Fig. 1. Percentage of 14C-Bayer 2353 remaining on thin layer 

plates, glass slides, and in an aqueous solution after exposure to long-wave UV light. Legend: = aqueous solution, = thin layer plates, and y = glass slides.

Table 4. Amounts of l4C-Bayer 2353 and l4C-degradation products from TLC plates spotted with an 

aqueous solution of 14C-Bayer 2353 exposed to longwave UV tight for up to 14 days (values expressed 

as percentages of total radioactivity from each 

plate)11.

Days 

and 

treatment

0

UV

Control

1

UV

Control

4

UV

Control

7

UV

Control

14

UV

Control

0

1

1

9

1

27

2

41

2

84

5

R/

0.13*> 0.34 0.41 0.65<=

- - - 99

- - - 99

- - - 91

- - - 99

- 4 3 66

- - - 98

- 6 4 49

- - - 98

7225

- - - 95

aSolution exposed to 9,800 ergs/s per cm2. 

bThese values are a composite of spots with R/s ranging from 

0.09 to 0.16. 

cThis R/ corresponds to that of authentic 14C-Bayer 2353.

Strufe and Gonnert (1962) are the only workers 

known to have attempted isolation or separation of 

degradation products/ of Bayer 73; they irradiated an 

ethanolic solution of the chemical for several days and 

then used paper chromatography to separate the 

products. Two compounds were found, in addition to 

the parent compound.

In our tests the 14C-Bayer 2353 was degraded by UV 

light into four to seven compounds.

References

Dawson, V. K. 1971. Photolytic sensitivity of Bayluscide. 

Quarterly report of progress, October 1971. U.S. Fish 

Wildl. Serv., Fish Control Lab., La Crosse, Wis. 27 pp.

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.

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

1964. Synergism of 5,2'-dichloro-4'-nitrosalicylanilide and 

3-trifluoromethyl-4-nitrophenol as a selective lamprey larvicide. Great Lakes Fish Comm., Tech. Rep. No. 8. 21 pp.

Meyling, A. H., C. H. J. Schutte, and R. J. Pitchford. 1962. 

Some laboratory investigations on Bayer 73 and ICI 

24223 as molluscicides. Bull. W.H.O. 27:95-98.

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

influence of environmental factors on the efficiency of 

Bayluscide. Pflanzenschutz-Nachr. 15:50-70.

Sturrock. R. F. 1974. Persistence of the molluscicide Bayluscide (clonitralide) emulsifiable concentrate on mud surfaces in the tropics. Ann. Trop. Med. Parasitol. 

68:427-434.

GPO 845-458

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

Invertebrates and Frog Larvae, by J. H. Chandler, Jr. 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. 1975.13 pp.

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

Lamprey, by G. W. Piavis and J. H. Howell. 1975.8 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. 1975.10 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. 

7pp.

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 R. 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. Cumming. 1976.13 pp.

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

T.D.Bills. 1976. llpp. 

(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. 5 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. 

(Reports 77 through 79 are in one cover.)

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 V. K. Dawson, 

K. B. Cumming, and P. A. Gilderhus. 1977.11 pp.

78. Toxicity of the Molluscicide Bayer 73 and Residue Dynamics of Bayer 2353 in Aquatic 

Invertebrates, by H. O. Sanders. 1977. 7 pp.

79. Accumulation, Elimination, and Biotransformation of the Lampricide 2',5-Dichloro-4'- 

nitrosalicylanilide by Chironomus tentans, by J. A. Kawatski and A. E. Zittel. 1977. 

8pp.

(Reports 80 and 81 are in one cover.)

80. Effects of Antimycin A and Rotenone on Macrobenthos in Ponds, by L. J. Houf and 

R. S. Campbell. 1977. 29 pp.

81. Aquatic Macroinvertebrates in a Small Wisconsin Trout Stream Before, During, and 

Two Years After Treatment with the Fish Toxicant Antimycin, by G. Z. Jacobi and 

D. J. Degan. 1977. 24 pp.

82. Investigations in Fish Control: Index to Numbers 1-72, 1964-76, by R. A. Schnick 

and K. A. Graves. 1977.19 pp.
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