INVESTIGATIONS IN FISH CONTROL

90. Accumulation and Loss of 2f,5-Dichloro-4f-nitrosalicylanilide 

(Bayer 73) by Fish: Laboratory Studies

91. Effects of Synergized Rotenone on Nontarget Organisms 

in Ponds

FISH * WILDLIFE 

SERVICE

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 National Fishery Research Laboratory at La Crosse, Wis., and the Southeastern Fish Control Laboratory at 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 inside back cover for list of current issues.]

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

90. Accumulation and Loss of 2',5-Dichloro-4'-mtrosalicylanilide 

(Bayer 73) by Fish: Laboratory Studies

By Verdel K. Dawson, Joe B. Sills, and Charles W. Luhning

91. Effects of Synergized Rotenone on Nontarget Organisms in Ponds

By R. M. Burress

UNITED STATES DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE 

Washington, D.C. 1982

Accumulation and Loss of 2 /,5-Dichloro-4'-nitrosalicylanilide 

(Bayer 73) by Fish: Laboratory Studies

by

Verdel K. Dawson

U,S. Fish and Wildlife Service 

National Fishery Research Laboratory

P. O. Box 818 

La Crosse, Wisconsin 54601

Joe B. Sills and Charles W. Luhning

U.S. Fish and Wildlife Service 

Southeastern Fish Control Laboratory

P. O. Box 9 

Warm Springs, Georgia 31830

Abstract

Residues of the 2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide (Bayer 73), as determined 

by gas-liquid chromatography, were rapidly accumulated by fish exposed to the lampricide in blood 

plasma, gallbladder bile, and muscle tissue of coho salmon (Oncorhynchus kistitch) and rainbow trout 

(Salmo gairdneri), and in bile and muscle of channel catfish (Ictalurus punctatus) and largemouth bass 

(Micropterus salmoides). Muscle residue levels increased to near the treatment concentration during 

exposure. After 10 days of withdrawal, residues in plasma, bile, and muscle of rainbow trout and coho 

salmon decreased to less than 1 % of their respective peak concentrations. In all four species, residues 

in muscle dropped below the limit of detection (0.01 jug/g) within 3 to 14 days. After an initial increase 

during early withdrawal, bile residues in all species declined steadily but had not dropped below initial 

levels in channel catfish in 14 days, or below detectable levels (0.01 jug/mL) in coho salmon after 28 

days of withdrawal.

The selective lampricide 3-trifluoromethyl-4-nitrophenol Materials and Methods 

(TFM) and the 2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide (Bayer 73) are used in combination (98% The study was organized in two parts: the determinaTFM, 2% Bayer 73) to control sea lampreys (Petromyzon tion of the rate of uptake during exposure to Bayer 73 and 

marinus) in tributaries of the Great Lakes. Treatment with the determination of the rate of withdrawal or elimination 

the combination has been shown to be more effective and after fish were transferred to lampricide-free water. Two 

less costly than treatment with TFM alone (Howell et al. coldwater and two warmwater species of fish were exposed 

1964). Because continued use of the mixture to control sea to 0.05 mg/L of technical grade (99.9%) Bayer 73 for 2, 

lampreys is subject to review by regulatory agencies, resi- 4, 8, 12, and 24 h to determine the rate of uptake of the 

due data from fish exposed to the lampricides will be needed lampricide. The coldwater species were treated in polyethyto renew registration. Accumulation and loss rates for TFM lene tanks containing 100 L of aerated well water (pH, 8.2; 

residues in fish were reported by Sills and Alien (1975), total alkalinity, 85 mg/L as CaCO3; total hardness, 100 mg/L 

D. C. G. Muir and W. L. Lockhart (unpublished report) asCaCO3)at!2 ± 1°C and the warmwater species in polydetermined the rate of uptake of 14C-niclosamide (Bayer 73 ethylene tanks containing 90 L of aerated, limed, spring 

without the ethanolamine salt) by fish. In the present study, water (pH, 7.3; total alkalinity, 25 mg/L as CaCO3 ; total 

we exposed four species of fish indigenous to Great Lakes hardness, 23.5 mg/L as CaCO3) at 19 ± 1°C. Coho salmon, 

tributaries to Bayer 73 and measured the concentration and Oncorhynchus kisutch, and rainbow trout, Salmo goirdpersistence of the lampricide in muscle, plasma, and bile. neri (average weights, 50 and 140 g, respectively), were
WITHDRAWAL TIME CDAYSJ

Fig. 1. Accumulation of Bayer 73 residues in plasma, bile, and 

muscle of rainbow trout during 12 h of exposure to 0.05 mg/L 

of the lampricide at 12 ± 1°C.

PLASMA

BILE

MUSCLE

10 12

EXPOSURE TIME CHOURS5

Fig. 2. Loss of Bayer 73 residues in plasma, bile, and muscle of 

rainbow trout exposed to 0.05 mg/L of the lampricide for 

12 h and then transferred to lampricide-free water at 12 ± 1°C.

treated and sampled at the National Fishery Research 

Laboratory, La Crosse, Wisconsin, and channel catfish, 

Ictalurw punctatus, and largemouth bass, Micropterns salmoidcfi (average weights, 280 and 330 g, respectively), at 

the Southeastern Fish Control Laboratory, Warm Springs, 

Georgia.

Five fish were exposed in each polyethylene tank. 

Samples of plasma, bile, and muscle tissue were taken from 

five rainbow trout at each sampling period in both the 

uptake and withdrawal studies. Muscle tissue was sampled 

for largemouth bass and channel catfish during the uptake 

stud\ and muscle and bile samples were taken from these 

two species and coho salmon during the withdrawal study 

in an attempt to identify species differences. Withdrawal 

studies \\cre conducted with fish that had been exposed to 

0.05 mu/L of Bayer 73 for 12 or 24 h, then transferred to 

lampricide-free water and sampled at selected time inter- 

\ als thereafter. Blood samples were drawn from the trout 

and salmon by puncture of the caudal peduncle. In collection of gallblader bile, fish were stunned by a blow to the 

head, a midventral incision was made, and bile was 

removed with needle and syringe.

Fish were not fed during exposure to the chemical, but 

food was provided after 24 h of withdrawal in fresh water. 

Because fish were fed during the withdrawal study, the 

volume of bile available for sampling was relatively small. 

Therefore the bile from the five fish in each sample group 

was pooled for analysis.

Frozen muscle samples were prepared for analysis by the 

method of Benville and Tindle (1970) and extracted by the 

column technique of Hesselberg and Johnson (1972). Extracts were processed and analyzed by the gas-liquid chromatographic method of Luhning et al. (1979). Blood plasma 

and bile samples were subjected to alkaline hydrolysis in 

the presence of hydrogen peroxide and analyzed for

Bayer 73 residues by gas-liquid chromatography (Luhning 

et al. 1979).

Results

Uptake Studies

Fish exposed to Bayer 73 rapidly accumulated the 

chemical, as demonstrated, for example, in rainbow trout 

(Fig. 1). Residues in muscle did not continue to increase 

with increasing exposure time, but rather plateaued and 

sometimes declined. The maximum residue concentration 

in muscle tissue of all species exposed was approximately 

that in the exposure solution (Tables 1 and 2).

The highest residue concentration observed in plasma of 

rainbow trout was 7.66 /xg/mL after 12 h of exposure, 

whereas residues in bile continued to increase throughout 

the uptake periods, to 473 /xg/mL at 24 h (Table 1).

Withdrawal Studies

Fish exposed to 0.05 mg/L of Bayer 73 for 12 or 24 h and 

then transferred to lampricide-free water eliminated residues accumulated during exposure. Residues in plasma, 

bile, and muscle of rainbow trout decreased to less than 

1 % of their respective peak concentrations within 10 days 

of withdrawal (Fig. 2). In muscle tissue, residues declined 

to less than detectable levels (<0.01 /xg/g) within 7 days 

in coho salmon, 10 days in rainbow trout, 3 days in channel 

catfish, and 14 days in largemouth bass (Tables 3-5). Elimination of residues in plasma to nondetectable levels required 

slightly longer 14 days in coho salmon and 21 days in rainbow trout (Tables 3 and 4).
Table 1. Mean concentrations of Bayer 73 in plasma 

and bile (ng/mL), and muscle (ng/g) from rainbow 

trout exposed to 0.05 mg/L of Bayer 73 for selected exposure times at 12 ± 1°C (SE in parentheses).

Exposure 

time (h) a

Control

2

4

8

12

24

Plasma

<0.01

2.36

(0.192)

2.96

(0.178)

5.84

(0.528)

7.66

(0.814)

5.30

(0.483)

Bile

<0.01

26.6

(10.9)

85.4

(17.7)

376.0

(81.4)

380.0

(50.1)

473.0

(36.7)

Muscle

<0.01

0.025

(0.008)

0.021

(0.003)

0.066

(0.004)

0.045

(0.008)

0.024

(0.004)

Table 2. Mean concentration of Bayer 73 (ng/g) in muscle 

tissue from channel catfish and largemouth bass exposed 

to 0.05 mg/L of Bayer 73 for selected exposure times at 

19 ± 1°C (SE in parentheses).

Exposure time (h) a

Control

2

4

8

12

24

Channel catfish

<0.01

0.051

(0.005)

0.034

(0.004)

0.025

(0.001)

0.022

(0.002)

0.019

(0.001)

Largemouth bass

<0.01

0.043

(0.010)

0.053

(0.008)

0.048

(0.007)

0.058

(0.003)

0.048

(0.003)

aFive fish were sampled at each interval.

aFive fish were sampled at each interval.

Residues in bile continued to increase for 12 to 24 h in 

coho salmon, rainbow trout, and channel catfish after the 

fish were transferred to fresh water, before they began

trending downward. The rate of elimination of residues 

from bile was similar to that in plasma and muscle in rainbow trout (Fig. 2), but because the peak concentrations in

Table 3. Mean concentrations of Bayer 73 in plasma and 

bile (fjLg/mL) and muscle (ng/g) from coho salmon exposed to 0.05 mg/L of Bayer 73 for 12 h and transferred 

to lampricide-free water at 12 ± 1° C for selected withdrawal times (SE in parentheses).

Withdrawal 

timea

Control

Hours

0

4

8

12

24

Days

3

7

10

14

21

28

Plasma

<0.01

5.00

(1.89)

8.48

(1.23)

6.24

(0.613)

6.57

(0.631)

5.47

(0.216)

1.80

(0.118)

0.439

(0.091)

0.073

(0.025)

<0.01

<0.01

<0.01

Bileb

<0.01

859

892

1,240

1,260

862

188

22.9

5.87

2.37

0.303

0.110

Muscle

<0.01

0.101

(0.006)

0.064

(0.005)

0.070

(0.008)

0.146

(0.031)

0.080

(0.005)

0.025

(0.004)

<0.01

<0.01

<0.01

<0.01

<0.01

Table 4. Mean concentrations of Bayer 73 in plasma and 

bile (/jLg/mL) and muscle (ng/g) from rainbow trout exposed to 0.05 mg/L of Bayer 73 for 12 h and transferred 

to lampricide-free water at 12 ± 1°C for selected withdrawal times (SE in parentheses).

Withdrawal 

time"

Control

Hours

0

4

8

12

24

Days

3

7

10

14

21

Plasma

<0.01

8.11

(2.29)

10.0

(1.72)

8.93

(0.921)

7.48

(0.747)

6.93

(1.27)

3.35

(0.997)

0.100

(0.013)

0.064

(0.011)

0.040

(0.003)

<0.01

Bile1 '

<0.01

726

313

1,000

792

1,180

117

0.95

1.45

1.11

0.76

Muscle

<0.01

0.146

(0.027)

0.130

(0.014)

0.141

(0.019)

0.118

(0.039)

0.160

(0.032)

0.022

(0.003)

0.010

(0.001)

<0.01

<0.01

<0.01

aFive fish were sampled at each interval. 

bPooled bile from five fish.

aFive fish were sampled at each interval. 

bPooled bile from five fish.
bile were so much higher, residues did not drop below detectable levels during the selected withdrawal periods in 

any of the species studied (Tables 3-5). Bile residues had 

not decreased from initial levels in channel catfish after 

14 days of withdrawal (Table 5) and were still detectable 

(0.11 /*g/mL) in coho salmon after 28 days of withdrawal 

(Table 3).

Residues in bile of the coldwater species exceeded 

1,100 /*g/mL (Tables 3 and 4), whereas the highest concentrations observed in the warmwater species were only 

54 and 126 /*g/mL (Table 5).

Discussion

The maximum observed muscle residue in all species 

exposed was approximately that of the exposure solution. 

The decline in muscle residues observed in rainbow trout 

and channel catfish during uptake exposures (Tables 1 

and 2) may have been a result of any one or a combination of the following factors: (1) biotransformation and 

excretion in the gallbladder bile (Alien et al. 1979; Statham 

and Lech 1975), (2) decreasing concentration in the exposure bath due to uptake or adsorption by fish, and (3) decomposition (Strufe and Gonnert 1962).

Muscle residues dissipated faster in channel catfish than 

in the other three species. Brown et al. (1971) suggested 

that scaleless fish may be capable of transporting chemicals across the integument, in addition to the gill membrane, thereby expediting uptake and elimination. Differences in the ratio of dark muscle to white muscle between 

open-water and bottom-dwelling species, and associated 

differences in chemical composition (Love 1970) and vascularization of the two muscle types (Satchell 1971), may 

also have contributed to the variations observed in rates of 

uptake and elimination.

From 3 to 14 days of withdrawal were required before 

residues of Bayer 73 in muscle dropped below 0.01 ^g/g. 

In comparison, Sills and Alien (1975) showed that only 

1 day was required for concentrations of TFM to fall below 

this level.

The continuing increase in bile residues for several hours 

after coho salmon and rainbow trout were transferred to 

fresh water, and its maintenance at initial or higher levels 

in channel catfish for 14 days, were not unexpected. Inasmuch as gallbladder bile is considered to be a major vehicle 

for collection, storage, and elimination of chemical residues (Lech et al. 1973), various residue pools within the 

organism may have continued to empty into the bile, even 

after the fish were transferred to fresh water. Food provided during the withdrawal study probably facilitated the 

mobilization of residues in the bile. The slow decrease in 

bile residues suggests that Bayer 73 may have been partly 

readsorbed and recycled from bile secreted into the digestive tract.

The method of analysis used for this study involves

Table 5. Mean concentrations of Bayer 73 in pooled 

samples of bile (pg/mL) and muscle (ng/g)from channel 

catfish and largemonth bass exposed to 0.05 mg/L of 

Bayer 73 for 24 h and transferred to lampricide-free 

water at 19 ± 1°C for selected withdrawal times (SE 

in parentheses).

Withdrawal 

time (days) 3

Control

0

1

3

7

10

14

Channel catfish

Bile1 '

NS

34.3

54.0

49.5

48.4

36.8

35.1

Muscle

<0.01

0.019

(0.002)

0.01

(0.001)

<0.01

<0.01

<0.01

<0.01

Largemouth bass

Bile1 '

NS

NS

NS

126

117

104

50

Muscle

<0.01

0.035

(0.002)

0.021

(0.002)

0.015

(0.000)

0.015

(0.003)

0.015

(0.002)

<0.01

"Five fish were sampled at each interval.

''Pooled bile from five fish; NS = no sample taken.

hydrolysis of Bayer 73 to form 2-chloro-4-nitroaniline, 

which is then analyzed by gas-liquid chromatography. Procedures for analysis of the methyl derivative of the intact 

molecule in water and sediment were described by Muir 

and Grift (1980). Fish have been reported to metabolize 

Bayer 73 by forming a glucuronide conjugate (Statham and 

Lech 1975; Schultz and Harman 1978; Alien et al. 1979). 

Although the hydrolysis procedure used in this study would 

break the glucuronide bond, the method for analysis of residues in muscle tissue measures only free Bayer 73 because 

the glucuronide conjugate, if present, is lost during the partitioning cleanup steps. Inasmuch as these steps were not 

used in the analysis of residues in plasma and bile, our 

values should accurately represent total residues in these 

fluids.

References

Alien, J. L., V. K. Davvson, and J. B. Hunn. 1979. Excretion of 

the lampricide Bayer 73 by rainbow trout. Pages 52-61 in L. L. 

Marking and R. A. Kimerle, eds. Aquatic toxicology. Proceedings of the second annual symposium on aquatic toxicology. 

American Society for Testing and Materials, ASTM Spec. Tech. 

Publ. 667.

Benville, P. E., Jr., and R. C. Tindle. 1970. Dry ice homogcnixation procedure for fish samples in pesticide residue analysis. J. 

Agric. Food Chem. 18:948-949.

Brown. E. A. B., J. E. Franklin, E. Pratt, and E. G. Trams. 1971. 

Contributions to the pharmacology of quinaldinc (uptake and 

distribution in the shark and comparative studies). (Symposium 

of elasmobranch biology Bar Harbor, Maine, June 20-23, 

1971.) Comp. Biochem. Physio!. 42:223-231.
Hesselberg, R. J., and J. L. Johnson. 1972. Column extraction of 

pesticides from fish, fish food, and mud. Bull. Environ. Contam. 

Toxicol. 7:115-120.

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 lampricide. Great Lakes 

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

Lech, J. J., S. K. Pepple, and C. N. Statham. 1973. Fish bile analysis: A possible aid in monitoring water quality. Toxicol. Appl. 

Pharmacol. 25:430-434.

Love, R. M. 1970. The chemical biology of fishes. Academic Press, 

New York. 547 pp.

Luhning, C. W., P. D. Harman, J. B. Sills, V. K. Dawson, and 

J. L. Alien. 1979. Gas-liquid chromatographic determination 

of Bayer 73 in fish, aquatic invertebrates, mud, and water. J. 

Assoc. Off. Anal. Chem. 62:1141-1145.

Muir, D. C. G., and N. P. Grift. 1980. Determination of niclosamide (Bayer 2353) in water and sediment samples. Int. J.

Environ. Anal. Chem. 8:1-14.

Satchell, G. H. 1971. Circulation in fishes. Cambridge University Press, New York. 131 pp.

Schultz, D. P., and P. D. Harman. 1978. Uptake, distribution, 

and elimination of the lampricide 2',5-dichloro-4'-nitro[ 14C]- 

salicylanilide (Bayer 2353) and its 2-aminoethanoI salt (Bayer 73) 

by largemouth bass. J. Agric. Food Chem. 26:1226-1230.

Sills, J. B., and J. L. Alien. 1975. Accumulation and loss of residues of 3-trifluoromethyl-4-nitrophenol (TFM) in fish muscle 

tissue: Laboratory studies. U.S. Fish Wildl. Serv., Invest. Fish 

Control 65. 10 pp.

Statham, C. N., and J. J. Lech. 1975. Metabolism of 2',5-dichloro4'-nitrosalicylanilide (Bayer 73) in rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can. 32:515-522.

Strufe, R., and R. Gonnert. 1962. Comparative studies on the influence of environmental factors upon the efficiency of Bayluscide. 

Pflanzenschutz-Nachr. 15:50-70.

Effects of Synergized Rotenone on Nontarget 

Organisms in Ponds

by

R. M. Burress

U.S. Fish and Wildlife Service 

Southeastern Fish Control Laboratory

P. O. Box 9 

Warm Springs, Georgia 31830

Abstract

Applications of 2- or 5-/xL/L concentrations of synergized rotenone (2.5%) in the Pro-Noxfish formulation to two shallow, 0.05-ha ponds caused a temporary reduction in both total numbers and diversity 

of benthic invertebrates and a total mortality of caged Asiatic clams (Corbicula manilensis) in both 

ponds, and a partial mortality of a resident population of larval leopard frogs (Rana pipiens) in the 

5-jtiL/L treatment. At day 7 after treatment, benthic organisms (no./m2) were reduced 67 % by the2-/xL/L 

concentration and 96% by the 5-/xL/L application. The diversity index (d) declined sharply in both 

ponds between days 3 and 7 after treatment, the lowest values being recorded on day 7 and day 37 

in the 2- and 5-/xL/L treatments, respectively. The equitability index (e) declined from day 3 to day 37 

in both ponds. By day 69, however, total numbers of benthic organisms had more than doubled over 

those originally present in the 2-/xL/L treatment (121 % increase), had more than tripled in the 5-/xL/L 

treatment (223% increase), and were virtually unchanged in the control pond (2% increase). Pretreatment zooplankton populations were low; no significant deleterious effects from the treatments were 

observed.

Formulations of rotenone have been used extensively to 

control fish populations. Concentrations of 0.5 to 1.0 /xL/L 

are adequate in most situations, but rotenone treatments 

from 0.1 to 5.0 /xL/L or more have been used, depending 

on the purpose of treatment, the fish species present, and 

conditions at the time of application (Schnick 1974). Chemical and biological conditions in eutrophic waters sometimes 

require use of the higher concentrations of rotenone (Orn 

1962; Spitler 1970). The labels of some rotenone products 

were recently amended to permit use of concentrations up 

to 5.0 /xL/L. The present study was conducted to evaluate 

the effects of 2.0- and 5.0-/xL/L concentrations of synergized rotenone on benthic invertebrates and zooplankters 

in ponds. The study was conducted in three experimental 

ponds at the Warm Springs (Georgia) National Fisji 

Hatchery from 23 August to 1 November 1976.

Methods

The rotenone formulation used was Pro-Noxfish, a synergized emulsifiable concentrate containing 2.5 % rotenone,

furnished by S. B. Penick & Co., New York, N.Y. This 

product contains 2.5% sulfoxide, a synergist used to enhance the activity of the rotenone.

The ponds used in this experiment have surface areas of 

about 0.05 ha, average and maximum depths of about 1.1 

and 1.5m, and clay bottoms. When checked before the start 

of the tests, each pond supported low populations of zooplankton, moderate populations of phytoplankton and 

benthic invertebrates, few aquatic plants, and no fish. 

About 2 weeks before treatment, applications of 20-20-5 

fertilizer and hydrated lime were added to each pond to 

stimulate plankton production, increase pH, and increase 

total hardness to about 20 mg/L.

On 24 August, ponds I and II were treated with 2- and 

5-/xL/L applications, respectively, and Pond III served as 

an untreated control. At the time of treatment, water temperatures were 31-32°C, dissolved oxygen concentrations 

were 8.5-11.5 mg/L (supersaturated), pH values were 

8.6-9.6, and Secchi disk transparencies were 43-80 cm 

(Table 1). Concentrations of rotenone (active ingredient) 

applied were 0.05 and 0.125 /iL/L. The required volumes 

of rotenone formulation were diluted in a tub filled with

1
pond water and then dispensed into the propeller wash of 

an outboard motor with a boat bailer.

Plankton and benthos samples were taken from each 

pond before treatment and at 3, 7, 14, 37, and 69 days after 

treatment. Sampling was terminated after 69 days because 

populations of most groups of benthic organisms appeared 

to have recovered from the treatments.

On each sampling date, duplicate samples of benthic 

organisms were taken from each pond by Ekman dredge 

(232 cm2 ) at 0.3,0.6,0.9,1.2, and 1.5m in each of the ponds 

(a total of 10 individually preserved samples from each 

pond). Samples were washed on a U.S. Standard No. 30 

sieve in the field and taken to the laboratory for further 

washing. The samples were stirred for a few minutes in a 

saturated aqueous solution of Epsom salts (magnesium sulfate) to facilitate separation of benthos from detritus. Floating organisms and low-density organic materials were then 

skimmed from the surface. This procedure was repeated 

at least three times and the remaining mixture of organisms 

and debris was preserved. Benthic organisms were removed 

and sorted into major taxa under a binocular microscope. 

I compared mean numbers of each taxon collected per grab 

on the several dates with pretreatment numbers, using Student's t test to determine whether observed declines in numbers were significant (P<0.05). Both mean diversity (d) and 

equitability (e) indices for the benthic invertebrates were 

calculated for each sampling day in each pond, according 

to methods outlined by Weber (1973). The major premise 

on which use of the indices is based is that relatively undisturbed environments support communities having large 

numbers of species, with no single species present in overwhelming abundance. Declines in d and e indices are often 

associated with adverse influences on benthic communities.

A battery-operated pump with a capacity of 16 L/min 

was used to collect zooplankton samples at depths of 0.3

and 1.2m. For each sample collection, 32 L of water were 

pumped through a Wisconsin plankton net (80-/tim mesh) 

as the boat was moved slowly about the ponds. Samples 

collected at each depth were concentrated and preserved 

in 10 mL of a solution consisting of 70 % ethyl alcohol, 20 % 

water, and 10% formalin. Numbers of cladocerans, copepods, and rotifers per liter of pond water were determined 

as suggested by Weber (1973). In addition, 100 adult Asiatic 

clams, Corbicula manilensis (10-14 mm long, 338-985 mg 

wet wt), were placed in a cage in each pond before treatment and observed for mortalities. Beginning 3 days after 

treatment, additional cages containing 20 Asiatic clams 

were placed in the treated ponds on each of 4 successive 

days. Clams that failed to close their valves or respond to 

stimuli were considered to be dead.

On the fourth day after treatment, 10 bluegills, Lepomis 

machrochirus (total length, 38-50 mm; weight, 2-4 g), 

were placed in a cage at a depth of 0.9 m in each pond. 

All fish were replaced each day until at least eight of the 

caged fish survived for 24 h.

Temperatures and concentrations of dissolved oxygen 

were determined at the surface and at 1.5 m on each sampling day. The Secchi disk transparency and the pH at the 

surface were also recorded in each pond to help ensure that 

mortalities of invertebrates did not result from adverse 

water quality.

Results and Discussion

Substantial phytoplankton blooms developed within 

3 days after treatment in ponds I and II, and transparency 

readings were reduced 50% or more (Table 1). Bonn and 

Holbert (1961) also observed an increase in the number of 

phytoplankters after the addition of rotenone to lake waters.

Table 1, Water quality measurements in ponds used to test effects of a synergized formulation of rotenone

(2.5% rotenone) on aquatic invertebrates.

Water quality measurements

Pond, treatment 

(/*L/L), and 

sampling day

Pond I, 2.0

0

3

7

Pond II, 5.0

0

3

7

Pond III, 0.0 (control) 

0

3

7

Temperature

Surface

31.0

30.7

28.0

31.9

32.5

27.8

31.0

30.3

28.0

Bottom

27.0

27.0

26.0

25.1

26.4

25.5

26.8

26.9

27.0

Dissolved oxygen

(mg/L)

Surface

11.5

13.2

>20.0

13.0

17.2

>20.0

8.5

10.1

17.2

Bottom

10.2

7.1

12.0

2.7

4.5

2.0

2.0

6.4

14.3

Secchi disk 

transparency 

(cm)

80

40

27

43

15

18

80

82

80
Table 2. Estimated total benthic invertebrates (no./m~) in bottom soil samples before and after treatment of pond I 

with 2 n.L/L of a synergized formulation of rotenone (2.5%).

Days after treatment

Taxa

Nematoda

Decapoda (Procambarus)

Ephemeroptera (Caenis)

Odonataa

Coleoptera

Agabus, Laccophilus

Dineutus

Berosus

Hemiptera

Notonecta

Hesperocorixa

Trichoptera (Oecetis)

Diptera

Chaoborus

Bezzia, Palpomyia

Glyptotendipes

Chrysops

Total organisms

0

0

0

766

4

13

4

211

17

30

56

254

56

3,862

0

5,273

3

0

0

159

13

69

0

603

116

82

39

164

112

2,644

0

4,001

7

0

0

4

4

47

0

56

0

13

0

125

13

1,507

0

1,769

37

17

0

202

22

13

0

715

4

34

0

4,585

116

8,284

0

13,992

69

525

9

474

0

26

0

577

17

0

0

3,475

422

6,118

4

11,647

aOdonata included five genera: Didymops, Erythemis, Ischnura, Libellula, and Ophiogomphtw,

They believed that the increase was caused by the reduction in numbers of plankton-feeding fishes, the increased 

food made available by decaying organic matter, and the 

additional depth of light penetration. By day 7, dissolved 

oxygen at the surface in all ponds had increased by more 

than 50%. The low concentrations of dissolved oxygen 

(2 mg/L) at the bottom in pond III on day 0 and in II on 

day 7 caused no mortalities among caged clams and were 

not considered to have been injurious to the benthic fauna.

All Asiatic clams died within 24 h after treatment in both 

treated ponds, and there was a substantial mortality of a 

resident population of larval leopard frogs (Rana pipiens) 

in pond II (5 /*L/L). However, among clams introduced 

into ponds I and II on the third day after treatment, none 

died within 25 days in pond I (2 /*L/L) and only three in 

II (5 fiL/L). No clams or bluegills died in the control pond 

during the study. Concentrations of rotenone were reduced 

to essentially nontoxic levels for clams by the fourth day 

after treatment, but among small bluegills placed in the 

treated ponds on the fourth day, mortality was 50% in 

pond I (2 pL/L) and 100% in pond II (5 pL/L). However, 

degradation proceeded rapidly and both the 2-/*L/L and 

5-/iL/L treatments were nontoxic to bluegills by the sixth 

day after treatment.

Populations of benthic invertebrates were similar at the 

outset in that all eight of the orders of organisms were found 

in all three ponds. The total number of invertebrate taxa 

initially collected in each pond ranged from 12 to 14, and 

the estimated total numbers of organisms varied widely 

among sample days (Tables 2, 3, and 4).

Pronounced changes in total numbers and diversity of 

benthic organisms occurred in both ponds after treatment, 

and many dead insect larvae were found in the 3- and 7-day 

samples. In pond I (2 fiL/L), the total number of organisms 

per unit of area was reduced by 66.5 % on day 7. No whirligig beetles (Dineutus), backswimmers (Notonecta), or 

caddisflies (Oecetis) were found, reducing the total number 

of orders of insects collected from six to five (Table 2). In 

contrast, Houf and Campbell (1977) reported that a 2-mg/L 

application of the Noxfish (5%) formulation of rotenone 

produced no immediate effects on abundance of benthic 

organisms in a shallow (0.6 m), mud-bottomed pond that 

was heavily vegetated. In their study, two factors mentioned by Lindgren (1960) may have contributed to the observed reduction in toxic effects: (1) the oxidation of rotenone in the vicinity of photosynthesizing vegetation; and 

(2) the sorption of rotenone by gyttja, clay, or gel-mud in 

the bottom soils. In pond II (5 jiL/L), the total numbers 

of organisms were reduced 96% by day 7; no nematodes, 

mayflies (Caenis), dragonflies (Libcllula, Ophiogomphis, 

Didymops, Erythemis, or Ischnura), caddisflies, or phantom midges (Chaohoru.s) were found, and the number of 

orders present dropped from six to two (Table 3). In pond 

III (control), there was also a pronounced drop in the total 

number of organisms present on day 7 (Table 4). Apparently, some of this reduction resulted from an emergence 

of chironomids (Glyptotendipes), whose numbers decreased 

by 48% during the preceding week.

Reductions in abundance of several taxa were observed 

at various intervals after treatment, but most were not
Table 3. Estimated total benthic invertebrates (no./rn2 ) in bottom soil samples before and after treatment of pond II 

with 5 n.L/L of a synergized formulation of rotenone (2.5%).

Davs after treatment

Taxa

Nematoda

Decapoda (Procambarus)

Ephemeroptera (Caenis)

Odonataa

Coleoptera

Agabus. Laccophihis

Dineittus

Berosus

Hemiptera (Hesperocorixa)

Trichoptera (Oecetis)

Diptera

Chaoborus

Bezzia, Palpomyia

Glyptotendipes

Total organisms

0

13

0

2,174

168

30

8

499

0

4

392

4

560

3,852

3

0

0

164

34

4

0

133

0

0

56

4

65

460

7

0

0

0

0

13

4

116

0

0

0

9

17

159

37

17

4

125

138

0

0

112

4

0

2,458

39

8,925

11,822

69

784

0

82

4

0

0

52

0

0

2,665

215

8,641

12,443

aOdonata included five genera: Didymops. Erythemis, Ischnura, LibeUula. and Ophiogomphus.

statistically significant. In pond I (2 /tL/L), a mortality of taken (Table 6). Although both populations increased theremayflies was readily evident, and significant reductions in after, the reduction in their numbers remained significant 

their numbers were observed on days 3, 7, and 37 (Table 5). on all later sampling dates until day 37, when numerous 

Inasmuch as water boatmen (Corixidae) were present in dragonflies were taken (82% of the original number). In 

only small numbers on the first four sampling dates, failure the control pond, the numbers of organisms in different taxa 

to collect them on day 69 was regarded as a sampling fluctuated widely, but no reductions were statistically siganomaly rather than a result of treatment. In pond II nificant (Table 7).

(5 /tL/L), mayflies and dragonflies were significantly Recovery from the effects of treatment varied among the

reduced on day 3; on day 7 no mayflies or dragonflies were different groups of organisms, but was especially rapid

Table 4. Estimated total benthic invertebrates (no./m") in bottom soil samples in pond III (control)

on days when treated ponds were sampled.

Days after treatment

Taxa 0 3 7 37 69

Nematoda

Decapoda

Procambarus

Palaemonetes

Ephemeroptera (Caenis)

Odonataa

Coleoptera (Berosus)

Hemiptera

Notonecta

Hesperoco rixa

Trichoptera (Oecetis)

Diptera

Chaoborus

Bezzia. Palpomyia

Glyptotendipes

Total organisms

22

0

73

0

9

4

4

0

0

1,085

99

17,110

18,406

17

0

73

0

13

0

0

17

0

288

43

11,814

12,265

99

0

164

0

0

4

0

22

0

2,316

146

8,852

11,603

56

4

69

4

4

4

13

0

4

112

336

7,552

8,158

263

0

103

0

4

9

4

0

0

4,994

633

12,731

18,741

aOdonata included five genera: Didymops, Erythemis, Ischnura, LibeUula, and Ophiogomphus.
Table 5. Mean numbers of numerically important groups of benthic invertebrates per grab (standard error of 

means in parentheses) and percent change from pretreatrnent numbers after treatment with 2 /j,L/L 

of a synergized formulation of rotenone (2.5%) in pond I.

Days after treatment

Pretreatment 3

Taxa

Ephemeroptera (Caenis)

Odonatab

Coleoptera

Agabm, Laccophilus

Berosus

Hemiptera

Notonecta

He.sperocorixa

Diptera

Chaoborus

Bezzia, Palpomyia

Ghjptotendipes

Total organisms

Mean 

number

17.8

(5.2)

0.1

(0.1)

0.3

(0.2)

4.9

(3.0)

0.4

(0.2)

0.7

(0.3)

5.9

(4.7)

1.3

(0.5)

89.7

(42.6)

121.1

(46.0)

Mean 

number

3.7a

(1.3)

0.3

(0.2)

1.6

(0.7)

17.6

(8.0)

2.7

(0.7)

1.9

(1.3)

3.8

(1.4)

2.6

(0.9)

61.4

(16.5)

95.5

(21.7)

Change

(%)

-79

200

433

259

575

171

-36

100

-32

-21

7

Mean 

number

O.l a

(0.1)

0.1

(0.1)

1.1

(0.6)

1.3

(0.6)

0.0

(0.0)

0.3

(0.3)

2.9

(2.2)

0.3

(0.2)

35.0

(8.0)

41.1

(7.2)

Change

(%)

-99

0

267

-73

-100

-57

-51

-77

-61

-66

3

Mean 

number

4.7a

(2.1)

0.5

(0.5)

0.3

(0.3)

16.6

(5.3)

0.1

(0.1)

0.8

(0.5)

106.5

(23.3)

2.7

(1.7)

192.4

(105.6)

.324.6

(112.3)

7

Change

(%)

-74

400

0

239

-75

14

1,705

108

114

168

69

Mean 

number

11.0

(3.9)

0.0

(0.0)

0.6

(0.3)

13.4

(5.2)

0.4

(0.2)

0.0*

(0.0)

80.7

(40.0)

9.8

(5.3)

142.1

(56.2)

245.8

(64.9)

Change

(%)

-38

-100

100

173

0

-100

1,268

654

58

103

aSignificant reduction in numbers from pretreatment samples.

bOdonata included five genera: Didymops, Enjthemis, Ischnura, Libellula. and Ophiogomphus.

among dipterans. The density of organisms (no./nT) in 

treated ponds on day 37 after treatment exceeded pretreatment populations by 165% in pond I and by 207% in II. 

In contrast, the numbers in the control pond on day 37 were 

56% less than before treatment. One month later (day 69), 

total numbers of organisms collected in each pond were 

greater than those before treatment by the following margins: 2-/iL/L treatment, 121 % ; 5-^L/L treatment, 223% ; 

and control, 2%. This finding corresponds with that of 

Cook and Moore (1969), who observed a rapid and explosive 

resurgence in insect numbers within 2 to 6 weeks after a 

late-summer rotenone treatment of a California creek.

Calculation of mean diversity (d) and equitability (e) 

indices for the benthic invertebrates showed that treatment 

caused a substantial decline in d values in both ponds I and 

II by day 7 (Table 8). In pond I (2 /xL/L), d had increased 

markedly by day 37, and was slightly higher than the_pretreatment value by day 69. In pond II, however, d on 

day 69 remained depressed but the dipteran population 

(largely Chaoborus and Ghjptotendipes} had increased so 

much that the total numbers of organisms per square meter 

greatly exceeded the numbers before treatment. In the control pond, d values fluctuated considerably, but were 

highest on day 69.

The e values in the control pond were consistently lower 

than those in the treated ponds (Table 8). The e values in 

both treated ponds were lowest on day 37, and the decline 

was greatest in the 5-/xL/L treatment. The reduction of both 

d and e values indicated that the rotenone treatments disturbed community structure, but a strong trend toward recovery was evident at 69 days after treatment.

Zooplankton populations in all three ponds remained 

consistently low throughout the study (Table 9). Minor fluctuations in abundance occurred, but the treatments did not 

produce the pronounced deleterious effects sometimes associated with rotenone applications (Almquist 1959; Kiser 

et al. 1963). Wollitz (1962) found that a treatment with 

0.95 /xL/L Pro-Noxfish in one experimental pond greatly 

reduced the zooplankton population, whereas a 0.70-/xL/L 

treatment in another pond apparently had little effect on 

most zooplankters. The reason for such differences is 

unclear. Kiser et al. (1963) suggested that the application 

of rotenone in spring and early summer, during zooplankton 

population pulses, appears to have more severe impacts and 

more lingering effects than applications made in autumn.
Table 6. Mean numbers of numerically important groups of benthic invertebrates per grab (standard error of 

means in parentheses) and percent change from pretreatment numbers after treatment with 5 nL/L of a synergized 

formulation of rotenone (2.5%) in pond II.

Pretreatment 3

Taxa

Nematoda

Ephemeroptera (Caenis)

Odonatab

Coleoptera

Agabus, Laccophilus

Berosus

Diptera

Chaoborus

Bezzia, Palpomyia

Glyptotendipes

Total organisms

Mean 

number

0.3

(0.2)

50.5

(9.6)

3.9

(1.3)

0.7

(0.4)

11.6

(5.9)

9.1

(5.7)

0.1

(0-1)

13.0

(9.5)

89.2

(18.0)

Mean Change 

number ( % )

0.0 -100

(0.0)

3.8a -92

(1.5)

0.8* -79

(0.4)

0.1 -86

(0.1)

3.1 -73

(1.9)

1.3 -86

(0.7)

0.1 0

(0.1)

1.5 -88

(0.6)

10.7 -88

(3-9)

Days after

7

Mean 

number

0.0

(0.0)

0.0a

(0.0)

0.0a

(0.0)

0.3

(0-2)

2.7

(1.2)

0.0

(0.0)

0.2

(0.2)

0.4

(0.2)

4.0

(1.2)

Change

(%)

-100

-100

-100

-57

-77

-100

100

-97

-96

treatment

37

Mean 

number

0.4

(0.2)

2.9a

(16.6)

3.2

(2.2)

0.0

(0.0)

2.6

(1.1)

57.1

(16.8)

0.9

(0.3)

207.3

(51.6)

274.5

(63.5)

Change

(%)

33

-94

-18

-100

-78

527

800

1,495

208

69

Mean 

number

18.2

(10.0)

1.9a

(0.6)

O.l a

(0.1)

0.0

(0.0)

1.2

(0.4)

61.9

(40.8)

5.0

(1.3)

200.7

(45.3)

289.0

(37.4)

Change 

(%)

5,967

-96

-97

-100

-90

580

4,900

1,444

224

Significant reduction in numbers from pretreatment samples.

bOdonata included five genera: Didymops, Erythemis, Ischnura, Libellula, and Ophiogomphus.

Table 7. Mean numbers of numerically important groups of benthic invertebrates per grab (standard error of means 

in parentheses) and percent change in numbers present in pond III (control) on various days after ponds I and II 

were treated.

Taxa

Nematoda

Odonata3

Coleoptera (Berosus)

Hemiptera (Notonecta)

Diptera

Chaoborus

Bezzia, Palpomyia

Glyptotendipes

Total organisms

Pretreatment

Mean 

number

0.5

(0.2)

0.2

(0.2)

0.1

(0.1)

0.1

(0.1)

25.2

(20.2)

2.3

(0.5)

397.4

(99.0)

425.8

(89.8)

3

Mean 

number

0.4

(0.3)

0.3

(0.2)

0.0

(0.0)

0.0

(0.0)

6.7

(3.0)

1.0

(0.4)

274.4

(82.2)

282.8

(62.7)

Change

(%)

-20

50

-100

-100

-73

-57

-31

-34

Days after

7

Mean 

number

2.3

(0.9)

0.0

(0.0)

0.1

(0.1)

0.0

(0.0)

54.8

(27.2)

3.4

(1.3)

205.7

(81.0)

266.3

(65.8)

Change

(%)

0

-100

0

-100

117

48

-48

-37

treatment

37

Mean 

number

2.3

(0.6)

0.1

(0.1)

0.1

(o.i)

0.3

(0.3)

2.6

(0-9)

7.8

(1.8)

175.4

(41.6)

187.9

(39.9)

Change

(%)

160

-50

0

200

-90

239

-56

-56

69

Mean 

number

6,1

(1.6)

0.1

(0.1)

0.2

(0.2)

0.1

(0.1)

116.0

(54.6)

14.7

(3.3)

295.7

(68.4)

432.9

(81.3)

Change

(%)

1,120

-50

100

0

360

539

-26

-38

aOdonata included five genera: Didymops, Erythemis, Ischnura, Libellula, and Ophiogomphus.
Table 8. Mean indices of diversity (d) and equitability (e) for benthic invertebrate samples collected at designated 

times from two ponds treated with a synergized formulation of rotenone and a control pond.

Concentration

no. (jiL/L)

I 2.0

II 5.0

III 0.0

(control)

Index

d

e

d

e

d

e

Pretreatment

1.6229

0.4885

1.8424

0.7771

0.4214

0.3039

3

1.8061

0.5038

2.1075

1.1455

0.2625

0.2647

Days after

7

0.8459

0.5177

0.5548

0.8419

0.9905

0.4734

treatment

37

1.3656

0.4550

1.0257

0.3484

0.4810

0.3286

69

1.6050

0.5511

1.2744

0.4924

1.1799

0.4360

Table 9. Estimated numbers of zooplankters per liter of water taken at selected intervals from pond III (control) and 

from ponds I and II, which were treated with 2.0- and 5.0-^L/L applications of rotenone, respectively.

Cladocera

Sampling day

Pretreatment

7

37

69

Pond I

0.47

0.00

0.00

0.00

Pond II

0.00

0.00

9.84

1.09

Pond III

0.00

0.00

3.44

0.16

Pond I

0.16

0.00

0.16

3.13

Copepoda

Pond II

0.16

0.16

0.16

2.19

Pond III

7.03

2.66

0.16

9.84

Pond I

0.00

0.63

0.00

0.31

Rotifera

Pond II

0.16

0.16

0.00

0.16

Pond III

0.00

0.00

0.00

2.03

References

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Rep. 40:146-160. 

Bonn, E. W., and L. R. Holbert. 1961. Some effects of rotenone

products on municipal water supplies. Trans. Am. Fish. Soc.

90:287-297.

Cook, S. F., Jr., and R. L. Moore. 1969. The effects of a rotenone treatment on the insect fauna of a California stream. Trans.

Am. Fish. Soc. 98:539-544. 

Houf, L. J., and R. S. Campbell. 1977. Effects of antimycin A

and rotenone on macrobenthos in ponds. U.S. Fish Wildl. Serv.,

Invest. Fish Control 80. 11 pp. 

Kiser, R. W., J. R. Donaldson, and P. R. Olson. 1963. The effect

of rotenone on zooplankton populations in freshwater lakes.

Trans. Am. Fish. Soc. 92:17-24.

Lindgren, P. E. 1960. About the effect of rotenone upon benthonic 

animals in lakes. Inst. Freshw. Res. Drottningholm Rep. 41: 

172-184.

Orn, B. 1962. Quantitative analysis of technical rotenone preparation. SodraSver. Fish for 1961-1962:26-38. (Water Pollut. 

Abstr. 39:374, 1966.)

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

rotenone in fisheries. U.S. Fish Wildl. Serv. Lit. Rev. 74-01. 

NTIS [Natl. Tech. Inf. Serv.] No. PB-235, 440/AS. 85 pp.

Spitler, R. J. 1970. An analysis of rotenone treatments for elimination of fish populations in southern Michigan lakes. 1957- 

1967. Mich. Acad. 3:77-82.

Weber, C. I., editor. 1973. Biological field and laboratory methods for measuring the quality of surface water and effluents. 

Environ. Monit. Ser. EPA [Environ. Prot. Agency]-670/4-73-001. 

38 pp.

Wollitz, R. E. 1962. Effects of certain commercial fish toxicants 

on the limnology of three cold-water ponds, Montana. Proc. 

Mont. Acad. Sci. 22:54-81.

GPO 836 - 407

(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 (Anodonto 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. 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. Gumming, 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. 8 pp. 

(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. 

(Reports 83 through 85 are in one cover.)

83. Survival of Two Species of Freshwater Clams, Corbicula leana and Magnonaias boykiniono, After Exposure to 

Antimycin, by L. L. Marking and J. H. Chandler, Jr. 1978. 5 pp.

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, and D. R. Buckler. 1978. 6 pp.

85. Hydrolysis and Photolysis of the Lampricide 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73), by D. P. Schultz and 

P. D. Harman. 1978. 5 pp.

86. Registration of Thirty-three Fishery Chemicals: Status of Research and Estimated Costs of Required Contract 

Studies, by R. A. Schnick and F. P. Meyer. 1978. 19 pp.

(Reports 87 through 89 are in one cover.)

87. Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an Anesthetic for Five Species of Freshwater Fish, by V. K. 

Dawson and P. A. Gilderhus. 1979. 5 pp.

88. Influences of Selected Environmental Factors on the Activity of a Prospective Fish Toxicant, 2-(Digeranyl-amino)- 

ethanol, in Laboratory Tests, by C. A. Launer and T. D. Bills. 1979. 4 pp.

89. Toxicities of the Lampricides 3-Trifluoromethyl-4-nitrophenol (TFM) and the 2-Aminoethanol Salt of 

2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) to Four Bird Species, by R. H. Hudson. 1979. 5 pp.
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.

NATIONAL FISHERY RESEARCH LABORATORY 

U.S. FISH AND WILDLIFE SERVICE

P.O. BOX 818 

LA CROSSE, WISCONSIN 54601

POSTAGE AND FEES PAID 

U.S. DEPARTMENT OF THE INTERIOR

INT 423