INVESTIGATIONS IN FISH CONTROL

96. Effects of Environmental Factors on the Toxicity of 

Chloramine-T to Fish

97. Effects of Organic Matter and Loading Rates of Fish 

on the Toxicity of Chloramine-T

UNITED STATES DEPARTMENT OF THE INTERIOR 

FISH AND WILDLIFE SERVICE
Investigations in Fish Control, published bv the Fish and Wildlife Service, include reports on the results of work 

at the National Fisheries Research Center at LaCrosse, Wisconsin, 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.

Copies of this publication may be obtained from the Publications Unit, U.S. Fish and Wildlife Service, 

Washington, DC 20240, or may be purchased from the National Technical Information Service (NTIS), 5285 

Port Royal Road, Springfield, VA 22161.

Library of Congress Cataloging in Publication Data

Effects of environmental factors on the toxicity of chloramine-T 

to fish.

(Investigations in fish control; 96-97)

Supt. of Docs, no.: I 49.70:96-97

1. Chloramine-T Toxicology. 2. Fishes Effect of drugs on. 

3. Chloramine-T Dose-response relationship. 4. ChloramineT Environmental aspects. I. Bills, T. D. II. Effects of organic 

matter and loading rates offish on the toxicity of chloramine-T. 

1988. III. Series. 

SH177.C39E44 1988 597'.024 88-600475
INVESTIGATIONS IN FISH CONTROL

96. Effects of Environmental Factors on the Toxicity of 

Chloramine-T to Fish

By T. D. Bills, L. L. Marking, V. K. Dawson, and J. J. Rach

97. Effects of Organic Matter and Loading Rates of Fish 

on the Toxicity of Chloramine-T

By T. D. Bills, L. L. Marking, V. K. Dawson, and G. E. Howe

U.S. Fish and Wildlife Service 

National Fisheries Research Center LaCrosse

P.O. Box 818 

LaCrosse, Wisconsin 54602

1988

Effects of Environmental Factors on the Toxicity of 

Chloramine-T to Fish

by

T. D. Bills, L. L. Marking, V. K. Dawson, 

and J. J. Rach

U.S. Fish and Wildlife Service 

National Fisheries Research Center

P.O. Box 818 

LaCrosse, Wisconsin 54602-0818

Abstract

The toxicity of chloramine-T (n-sodium-n-chloro-para-toluenesulfonamide), a therapeutant used 

to treat bacterial gill disease in fish, was evaluated under a variety of physical and chemical conditions. The toxicity (96-h LC50) was 2.80 mg/L for rainbow trout, Salmo gairdneri; 3.75 mg/L 

for channel catfish, Ictalurus punctatus; and 7.30 mg/L for fathead minnows, Pimephales promelas. 

Chloramine-T was more toxic in warm water than in cold water at exposures of 24 h or less, but 

temperature had no significant effect on toxicity in 96-h exposures. The chemical was slightly less 

toxic to rainbow trout in hard than in soft water, but water hardness had little influence on its toxicity to channel catfish. The pH was the most important factor affecting chloramine-T toxicity; the 

chemical was about 6 times more toxic at pH 6.5 than at 9.5. At levels X1, X3, and X5 the recommended use concentration of 12 mg/L for 1 h, there was no significant mortality in rainbow trout 

and no abnormal responses were observed in treated fish. In rainbow trout, the toxicity of solutions 

of chloramine-T aged for 4 weeks was about half that of fresh solutions (deactivation index = 2.10).

Introduction Researchers have reported that the activity of

	chloramine-T is affected by pH, water hardness, and

Chloramine-T (n-sodium-n-chloro-para-toluenesulfona- temperature. Tooby et al. (1975) found that the toxicity

mide) was shown by From (1980) to be effective for con- of chloramine-T to fish and eggs increased as pH and

trolling bacterial gill disease (BGD), which is one of the water hardness decreased. Cross and Hursey (1973)

most common diseases of hatchery-reared salmonids and observed that chloramine-T was more toxic to fish in soft,

causes more fish losses than any other bacterial disease, acid waters than in hard, alkaline waters, and less toxic

The disease is strongly mediated by the stressful environ- at 10° C than at high temperatures,

mental conditions and marginal nutrition commonly The purpose of the present study was to delineate the

associated with intensive culture. Flavobacteria and flexi- five factors that affect the toxicity of chloramine-T to

bacteria are associated agents (Snieszko 1981). Gills selected species of fish: (1) sensitivity differences between

damaged by BGD pathogens are also prone to secondary selected coldwater and warmwater fish species; (2) efinvasion by fungi (Warren 1981). Because BGD is a limit- fects of pH, water hardness, and temperature; (3) pering factor in fish production, a control agent, such as sistence of toxicity in solutions over a 4-week period;

chloramine-T, is needed for use on food fish. Approval (4) toxicity and observed effects at recommended use

by the U.S. Food and Drug Administration will be pattern concentrations for rainbow trout (Salmo gairdrequired. neri)\ and (5) the time-independent toxicity.

	1
Materials and Methods

Static and flow-through test procedures used in this 

study followed those prescribed by the Committee on 

Methods for Toxicity Tests with Aquatic Organisms 

(1975), ASTM Committee E-35 on Pesticides (1980), and 

U.S. Department of Agriculture (1986). We exposed 

20 fish to each concentration of chloramine-T in glass jars 

containing 15 L of oxygen-saturated test water. Reconstituted test waters were prepared according to standardized procedures to produce the desired water quality. The 

pH of test waters was controlled with chemical buffers 

(Committee on Methods for Toxicity Tests with Aquatic 

Organisms 1975). The solutions were adjusted to the 

desired pH before each test began and readjusted with 

chemical buffers at 24-h intervals, as needed, to maintain the selected pH (±0.2 unit). Temperatures were regulated by immersing the test jars in constant temperature 

water baths. To assess the effects of water hardness, we 

buffered solutions to a constant pH with sodium bicarbonate using the procedure of Marking (1975).

Rainbow trout, fathead minnows (Pimephales promelas), and channel catfish (Ictalurus punctatus), obtained from a Federal fish hatchery or produced at the 

National Fisheries Research Center, LaCrosse, Wisconsin, were maintained according to the standard procedures for handling bioassay fish described by Hunn et al. 

(1968). The fish were acclimated to the desired water 

chemistries and temperatures for 24 h before each test. 

Mortalities were recorded at 1, 3, 6, 12, 24, 48, 72, and 

96 h.

Two species (rainbow trout and channel catfish) were 

used in tests to determine the effects of water temperature, hardness, and pH on the toxicity of chloramine-T. 

In tests on the effects of use pattern levels, we exposed 

rainbow trout to chloramine-T (12 mg/L for 1 h) and to 

x3 and x5 levels, as prescribed in the IR-4 Guidelines 

for Investigation of Minor Use Drugs (U.S. Department 

of Agriculture 1986). We observed responses offish after 

exposure for 14 days, using the criteria of Lennon and 

Walker (1964).

Commercial grade chloramine-T (lot 12605), obtained 

from Badger Pharmacal Inc. (Jackson, Wisconsin), was 

accompanied by a certificate of analysis that listed the 

assay as 100.03% available chlorine. When we tested the 

material, using the method provided by the manufacturer 

(U.S. Pharmacopeia! Convention, Inc. 1979), the material 

yielded 98.4% available chlorine.

Concentrations of chloramine-T in test waters were 

determined by high performance liquid chromatography 

(HPLC) at 0, 6, 24, 48, 72, and 96 h. Samples were

filtered through 0.45 /urn Acrodiscs, and then injected 

directly onto the column with an automatic Waters 

Intelligent Sample Processor (WISP). Retention time was 

about 3.2 min. Quantification of the peaks was performed by a 730 Data Module having external standard 

calibration.

The Waters Associates, Inc., high performance liquid 

chromatography unit that we used consisted of a Model 

481 Lambda-Max LC spectrophotometer, Model 510 

pump, Model 710B WISP auto sampler, and 730 Data 

Module. The operating conditions were as follows: stationary phase, 30 cm x 4 mm Varian MicroPak MCH-10; 

mobile phase, acetonitrile: phosphate buffer (50:50, v/v); 

flow rate, 2.0 mL/min; chart speed, 2.0 cm/min; wavelength, 229 nm; and attenuation, 0.10 absorption units. 

A 0.45-jnm disposal filter assembly was used to filter the 

sample.

Reagents were acetonitrile, HPLC grade; water, HPLC 

grade; phosphoric acid, American Chemical Society reagent grade 0.2 M, 13.6 mL diluted to 1 L with HPLC 

water; monobasic potassium phosphate, American Chemical Society reagent grade 0.2 M, consisting of 27.2 g 

of KH2PO4 diluted to 1 L with HPLC water; and buffer 

reagent, 0.1 M, consisting of 14.3 mL of 0.2 M H3PO4 

+ 10.7 mL of 0.2 M KH2PO4 diluted to 500 mL (pH 

= 2.6).

In tests to determine the persistence of chloramine-T 

in water, we aged solutions in glass containers under 

routine laboratory conditions with 12-h photoperiods 

for as long as 4 weeks. Residual concentrations of 

chloramine-T in the aged solutions were determined analytically each week and the toxicity was compared with 

that of fresh solutions. Deactivation indices were calculated by dividing the 96-h LC50 of the aged solutions by 

the 96-h LC50 of the fresh solutions (Marking 1972).

The methods of Litchfield and Wilcoxon (1949) were 

used to compute the LC50's and 95% confidence intervals. Time-independent LC50's (TILC50) were calculated 

according to the method of Green (1965).

Results and Discussion 

Toxicity to Selected Species of Fish

Chloramine-T was toxic to all species exposed in soft 

water; the 96-h LC50's (mg/L) were 2.80 for rainbow 

trout, 3.75 for channel catfish, and 7.30 for fathead minnows (Table 1). Rainbow trout (coldwater) and channel 

catfish (warmwater) were thus twice as sensitive as fathead minnows (warmwater).
Table 1. Toxicity of chloramine-T to three species offish in soft water at 12 ° C.

Species

Rainbow trout

Fathead minnow

Channel catfish

Lot

8640

8631

8632

Source3

Erwin NFH

LaCrosse NFRC

LaCrosse NFRC

Mean weight

(g)

0.73

1.11

1.50

96-h LC50 and 95%

confidence interval

(mg/L)

2.80

2.41-3.26

7.30

6.71-7.94

3.75

3.30-4.26

aNFH = National Fish Hatchery; NFRC = National Fisheries Research Center.

Influence of Temperature, Water mortality in 3 h at 60 mg/L at any temperature (Table 3).

Hardness and nH ^er 6 h at temperatures of 22, 17, and 12° C, the LCSO's

	were 14.0, 25.3, and >60.0 mg/L, respectively. TemThe toxicity of chloramine-T was affected by water perature apparently affected the time of response rather

characteristics, and alterations in toxicity were consistent than the ultimate toxicity of chloramine-T; the 96-h

between the two species. The chemical was more toxic LCSO's did not differ significantly for either species,

in warm water than in cold water in exposures of 24 h Water hardness had only a slight effect on the toxor less. For example, the 3-h LC50 for rainbow trout was icity of chloramine-T to rainbow trout and no effect

28.0 mg/L at 17° C and 43 mg/L at 12° C; at 7° C there on channel catfish (Tables 2 and 3). For rainbow trout,

was no mortality at 60 mg/L (Table 2). Channel catfish the 96-h LC50 was 7.35 mg/L in very soft water

did not respond as quickly to the chemical; there was no (10-13 mg/L as CaCOs) and 14.2 mg/L in very hard

Table 2. Toxicity (LC50, mg/L and 95% confidence interval) of chloramine-T to rainbow trout in water of different

temperatures, water hardnesses, and pH levels.

Duration of test (h)

Temperature 

(°C) Hardness pH 1 3 6 12 24 96

7

12

17

12

12

12

12

12

12

12

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

7.5

7.5

7.5

8.1

8.1

8.1

8.1

6.5

8.5

9.5

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

55.8

48.4-64.3

>60.0

>60.0

>60.0

43.0

36.9-50.1

28.0

22.7-34.5

>60.0

>60.0

>60.0

>60.0

13.5

11.2-16.2

>60.0

>60.0

28.0

22.8-34.4

17.5

14.8-20.7

10.0

9.07-11.0

>60.0

>60.0

>60.0

>60.0

8.22

7.71-8.76

>60.0

>60.0

18.0

15.3-21.1

14.0

11.3-17.3

8.00

7.57-8.46

38.7

33.9-44.2

47.5

 

45.0

38.5-52.6

49.0

 

6.05

5.17-7.08

55.7

49.1-63.1

>60.0

11.7

10.5-13.0

6.90

6.31-7.54

4.90

4.48-5.35

20.2

17.7-23.0

25.4

21.0-30.6

25.0

20.7-30.1

25.5

21.2-30.7

2.81

2.41-3.27

46.0

39.5-53.6

>60.0

4.30

3.69-5.01

2.80

2.41-3.26

2.80

2.27-3.46

7.35

6.92-7.80

9.00

9.33-9.72

10.0

9.04-11.1

14.2

12.2-16.5

1.89

1.63-2.19

11.0

9.78-12.4

10.8

9.96-12.2
Table 3. Toxicity (LC50, mg/L and 95% confidence interval) of chloramine-T to channel catfish in water of selected

temperatures, water hardnesses, and pH levels.

Temperature

(°C) Hardness pH

Duration of test (h)

12 24 96

12

17

22

12

12

12

12

12

12

12

Soft

Soft

Soft

Very soft

Soft

Hard

Very hard

Soft

Soft

Soft

7.5

7.5

7.5

8.1

8.1

8.1

8.1

6.5

8.5

9.5

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

>60.0

27.0

21.0-35.0

>60.0

>60.0

>60.0

25.3

21.0-30.4

14.0

12.0-16.3

>60.0

>60.0

>60.0

>60.0

10.0

9.07-11.0

>60.0

>60.0

14.2

12.2-16.5

14.2

12.2-16.5

9.00

8.51-9.51

>60.0

>60.0

>60.0

>60.0

5.60

5.21-6.01

>60.0

>60.0

10.0

9.07-11.0

7.20

6.78-7.64

5.63

5.08-6.24

28.0

24.1-32.6

30.5

26.3-35.4

33.0

29.2-37.3

37.0

32.7-41.8

2.85

2.52-3.22

51.5

46.8-56.7

>60.0

3.75

3.30-4.26

3.73

3.29-4.22

3.80

3.33-4.34

7.70

6.98-8.49

11.0

9.13-13.2

7.80

6.53-9.31

9.80

8.64-11.1

1.75

1.48-2.06

10.5

9.14-12.1

12.3

9.75-15.5

water (280-320 mg/L as CaCO3). Tooby et al. (1975) 

and Cross and Hursey (1973) reported that chloramine-T 

was more toxic in soft, acid waters than in hard, alkaline 

waters, but they did not isolate the effects of hardness from 

those of pH.

The toxicity of chloramine-T increased significantly as 

the pH of test waters decreased for both species (Tables 2 

and 3). At pH 9.5, no rainbow trout or channel catfish 

died after 24 h of exposure to 60 mg/L, but the chemical 

was about 6 times more toxic to both species at pH 6.5. 

The 96-h LC50's for rainbow trout and channel catfish 

were 1.89 and 1.75 mg/L in acid water (pH 6.5) compared with 10.8 and 12.3 mg/L in alkaline water (pH 9.5), 

respectively (Tables 2 and 3).

Use Pattern Exposure

Responses of rainbow trout exposed to chloramine-T 

for 1 h to x 1, x3, and x 5 the recommended use pattern 

concentration (12 mg/L) did not differ from those of control fish during the 14-day posttreatment recovery period. 

These fish were exposed in soft water at 12° C and 

pH 7.5. Therefore, there was little chance of causing mortality with overtreatment.

Persistence

The toxicity of aged solutions of chloramine-T decreased with aging (Table 4). Toxicity to rainbow trout 

was decreased by a factor of 1/2 after 4 weeks; the 96-h

Table 4. Toxicity and deactivation index for chloramine-T 

for rainbow trout in soft water at 12 ° C.

Aging period 

(weeks)

0

1

2

3

4

96-h LC50 (mg/L) 

and 95% confidence 

interval

2.85

2.57-3.16

4.10

3.54-4.72

3.80

3.34-4.32

8.00

6.15-10.4

6.00

5.27-6.82

Deactivation 

index3

1.0

1.44

1.33

2.81

2.10

aLC50 of aged solution 

LC50 of fresh solution
Table 5. HPLC analysis of chloramine-T concentrations of 6, 10, and 40 mg/L (calculated) remaining in soft water

without fish at 12° C for periods as long as 4 weeks.

Age of solution 

at sampling tin 

(weeks)

0

1

2

3

4

le 

6

6.12

5.82

5.43

5.41

5.50

Calculated concentration (mg/L)

10

10.26

9.82

9.34

9.33

9.56

40

40.59

39.60

37.48

38.98

40.81

6

5.84

5.44

5.62

5.58

9

9

9

9

10

.88

.37

.38

.66

40

39.33

37.46

39.22

41.11

6

5.78

5.76

5.89

10

9.58

9.73

9.87

40 6 10 40 6

37.94 5.88 9.87 39.60 5.94

39.06 5.83 9.85 39.75

40.29

10 40

10.02 39.65

LC50 was 6.00 mg/L, in comparison with 2.85 mg/L for 

freshly prepared solutions (deactivation index = 2.10). 

In analytical checks of the concentrations of chloramine-T 

made at weekly intervals throughout the aging experiment, 

the measured concentrations (Table 5) did not decrease 

as rapidly as the toxicity of the aged solutions. For example, the 6 mg/L (calculated) concentration was 

measured at 6.12 mg/L at time 0 and 5.50 mg/L after 

4 weeks of aging. For the same aging period, the toxicity decreased by a factor of 2. When calculated exposure 

concentrations of 6, 10, and 40 mg/L were analyzed by 

HPLC at the beginning of all tests to verify the accuracy 

of the chemical additions, the means and standard deviations (mg/L) for the three analyzed concentrations from 

40 tests were 5.90+0.17, 9.89+0.27, and 38.9 + 1.39.

Time-independent Toxicity

Rainbow trout exposed to chloramine-T in flow-through 

tests were considerably more resistant than those exposed 

in static tests. In two separate exposures, the 96-h LCSO's 

were 23.0 and 30.0 mg/L in flow-through tests and 7.0 

and 4.6 mg/L in static tests (Table 6). We speculate that 

chlorine is released from chloramine-T more completely 

under static conditions and believe that this increased 

release causes the higher toxicity. Mortalities offish exposed to chloramine-T did not continue beyond 96 h; the 

time-independent LCSO's were similar to the 96-h LCSO's 

in flow-through toxicity tests (Table 6).

References

ASTM Committee E-35 on Pesticides. 1980. Standard practice 

for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians, E729-80. Pages 1-25 in Annual 

book of ASTM standards, Part 46. End use and consumer 

products. American Society for Testing and Materials, 

Philadelphia, Pa.

Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. Environ. Prot. Agency, 

Ecol. Res. Serv., EPA-660/3-75-009. 61 pp.

Cross, D. G., and P. A. Hursey. 1973. Chloramine-T for the 

control of Ichthyopthirius multifilis (Fouquet). J. Fish Biol. 

5:789-798.

From, J. 1980. Chloramine-T for control of bacterial gill disease. 

Prog. Fish-Cult. 42:85-86.

Green, R. H. 1965. Estimation of tolerance over an indefinite 

time period. Ecology 46:887.

Hunn, J. B., R. A. Schoettger, and E. W. Whealdon. 1968. 

Observations on handling and maintenance of bioassay fish. 

Prog. Fish-Cult. 30:164-167.

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

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

Serv., Invest. Fish Control 1, Circ. 185. 15 pp.

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

method of evaluating dose-effect experiments. J. Pharmacol. 

Exp. Ther. 96:99-113.

Table 6. Acute and chronic (time-independent) toxicity 

of chloramine-T to rainbow trout in well water at 12 ° C.

LCSO's (mg/L) and 95% 

confidence intervals

nsn source anu 

type of test

Lot 8675

Static

Flow-through

Lot 8706

Static

Flow-through

96 h

4.60

4.14-5.11

30.0

27.6-32.6

7.00

6.44-7.61

23.0

20.2-26.2

TILC50a

 

24.3

21.5-27.5

 

23.0

20.1-26.2

aTime-independent LC50.
Marking, L. L. 1972. Methods of estimating the half-life of 

biological activity of toxic chemicals in water. U.S. Fish 

Wildl. Serv., Invest. Fish Control 46. 9 pp.

Marking, L. L. 1975. Toxicological protocol for the development of piscicides. Pages 26-31 in P. H. Eschmeyer, ed. 

Rehabilitation of fish populations with toxicants: A symposium. Am. Fish. Soc. Spec. Publ. 4.

Snieszko, S. F. 1981. Bacterial gill disease of freshwater fishes. 

U.S. Fish Wildl. Serv., Fish Dis. Leafl. 62. 11 pp.

Tooby, T. E., P. A. Hursey, and J. S. Alabaster. 1975. The 

acute toxicity of 102 pesticides and miscellaneous substances

to fish. Chem. Ind. (21 June 1975):523-526. 

U.S. Department of Agriculture. 1986. Interregional research

project no. 4. Guidelines for IR-4 investigations. New Jersey

Agricultural Experiment Station, Cook College, Rutgers

University, New Brunswick, N.J. 6pp. 

U.S. Pharmacopeial Convention, Inc. 1979. Chloramine-T

assay. Pages 1057-1058 in U.S. Pharmacopia. 20th Revision.

Mack Publishing Co., Easton, Pa. 

Warren, J. W. 1981. Diseases of hatchery fish. U.S. Fish and

 Wildlife Service, Twin Cities, Minn. 91 pp.
Bills, Terry D., Leif L. Marking, Verdel K. Dawson, and Jeffery J. Rach. 1988. 

Effects of environmental factors on the toxicity of chloramine-T to fish.

U.S. Fish Wildl. Serv., Invest. Fish Control 96. 6 pp.

The toxicity of chloramine-T (n-sodium-n-chloro-para-toluenesulfonamide), 

a therapeutant used to treat bacterial gill disease in fish, was evaluated under 

a variety of environmental conditions. The 96-h LC50 (mg/L) was 2.80 for rainbow trout, Salmo gairdneri; 3.75 mg/L for channel catfish, Ictaluruspunctatus; 

and 7.30 mg/L for fathead minnows, Pimephales promelas. Chloramine-T was 

more toxic in warm water than in cold water only at exposures of 24 h or less; 

it was about 6 times more toxic at pH 6.5 than at 9.5, but nontoxic at levels 

x5 the use pattern level (12 mg/L; 1 h), and detoxified to half the original activity 

in about 4 weeks.

Key words: Toxicity, chloramine-T, pH, water hardness, temperature.

Bills, Terry D., Leif L. Marking, Verdel K

Effects of environmental factors on t

U.S. Fish Wildl. Serv., Invest. Fish C

The toxicity of chloramine-T (n-sodium

a therapeutant used to treat bacterial gill 

a variety of environmental conditions. The 

bow trout, Salmo gairdneri; 3.75 mg/L fo

and 7.30 mg/L for fathead minnows, Pim

more toxic in warm water than in cold wa

it was about 6 times more toxic at pH 6

x5 the use pattern level (12 mg/L; 1 h), an

in about 4 weeks.

Key words: Toxicity, chloramine-T, pH

Bills, Terry D., Leif L. Marking, Verdel K. Dawson, and Jeffery J. Rach. 1988. Effects of environmental factors on the toxicity of chloramine-T to fish. U.S. Fish Wildl. Serv., Invest. Fish Control 

96. 6 pp.

The toxicity of chloramine-T (n-sodium-n-chloro-para-toluenesulfonamide), a therapeutant used to 

treat bacterial gill disease in fish, was evaluated under a variety of environmental conditions. The 96-h 

LC50 (mg/L) was 2.80 for rainbow trout, Salmo gairdneri; 3.75 mg/L for channel catfish, Ictalurus 

punctatus; and 7.30 mg/L for fathead minnows, Pimephales promelas. Chloramine-T was more toxic 

in warm water than in cold water only at exposures of 24 h or less; it was about 6 times more toxic 

at pH 6.5 than at 9.5, but nontoxic at levels x5 the use pattern level (12 mg/L; 1 h), and detoxified 

to half the original activity in about 4 weeks.

Key words: Toxicity, chloramine-T, pH, water hardness, temperature.

Bills, Terry D., Leif L. Marking, Verdel K. Dawson, a

tal factors on the toxicity of chloramine-T to fis

96. 6 pp.

The toxicity of chloramine-T (n-sodium-n-chloro-p

treat bacterial gill disease in fish, was evaluated under 

LC50 (mg/L) was 2.80 for rainbow trout, Salmo gai

punctatus; and 7.30 mg/L for fathead minnows, Pime

in warm water than in cold water only at exposures 

at pH 6.5 than at 9.5, but nontoxic at levels x5 the 

to half the original activity in about 4 weeks.

Key words: Toxicity, chloramine-T, pH, water har

Effects of Organic Matter and Loading Rates of Fish 

on the Toxicity of Chloramine-T

by

T. D. Bills, L. L. Marking, V. K. Dawson, and G. E. Howe

U.S. Fish and Wildlife Service 

National Fisheries Research Center

P.O. Box 818 

LaCrosse, Wisconsin 54602-0818

Abstract

Chloramine-T («-sodium-«-chloro-para-toluenesulfonamide) is effective for the control of bacterial 

gill disease in fishes but data on its toxicity and safety are lacking. We examined the effect of fish 

loading rates, feed levels, and fecal material on the availability and toxicity of the chemical. Its 

toxicity increased (24-h LC50's were 36 mg/L and 14 mg/L) as loading rates of fathead minnows 

(Pimephales promelas) increased from 0.52 to 2.07 g/L of water. The presence of fish food decreased the toxicity of the chemical. The 96-h LC50 was 7.30 mg/L without feed and 14.2 and 

20.8 mg/L after the addition of 0.07 and 0.20 g/L of fish food. The presence of fecal material also 

significantly decreased the toxicity of chloramine-T. Concentrations in water decreased more rapidly 

when fish were present than when only feed or fecal matter was present. The decreases were greatest 

in the presence of fish and feed or fish and fecal material.

Introduction

Chloramine-T (w-sodium-w-chloro-para-toluenesulfonamide) is an antibacterial agent used as a disinfectant and 

preservative in the food industry. It is also effective for 

controlling bacterial gill disease (BGD) in fish (From 

1980). Although no single pathogen seems to be responsible for BGD infections, all known agents are gramnegative bacteria myxobacteria, aeromonads, and 

pseudomonads (Snieszko 1981). The disease is highly contagious among cultured salmonids and is associated with 

crowded rearing conditions and sometimes inadequate 

nutrition. The disease can lead to substantial fish losses. 

An approved therapeutant to control BGD is needed to 

enable the production of salmonids for the restoration of 

fish stocks and for the sport and commercial fisheries.

Several researchers have reported that the activity of 

chloramine-T is affected by environmental factors. Van 

Duijn (1967) reported that it is partly or totally inactivated by organic detritus. Similarly, Cross and Hursey

(1973) demonstrated that chloramine-T lost its activity in 

the presence of excessive organic matter and showed that 

its toxicity to fish was greater in soft, acid waters than 

in hard, alkaline waters. Bills et al. (1988) reported that 

the acute toxicity of chloramine-T was greater at a given 

concentration in warm than in cold water and that the compound was more toxic in acid than in alkaline water. 

The purpose of the present study was to determine

(1) the effects offish loading rates, feed levels, and fecal 

material on the toxicity of chloramine-T to fish; and

(2) the available concentration of chloramine-T in water 

containing fish, feed, and fecal material.

Materials and Methods

Static test procedures used in this study followed those 

prescribed by the Committee on Methods for Toxicity 

with Aquatic Organisms (1975), ASTM Committee E-35 

on Pesticides (1980), and U.S. Department of Agriculture

1
(1986). Reconstituted test water (total hardness, 

40-44 mg/L as CaCC^) was prepared according to 

standardized procedures. Glass jars containing 15 L of 

oxygen-saturated water were used for all tests. Exposure 

temperatures were regulated by immersing the test jars 

in constant temperature water baths at 12° C. All tests 

were conducted in duplicate. Twenty fish were exposed 

to each concentration, except in the fish-loading rate 

study, in which 7, 14, or 28 fish were used.

Fathead minnows (Pimephales promelas) cultured at the 

National Fisheries Research Center, LaCrosse, Wisconsin, were used as the test organisms. They were maintained according to the standard procedures for handling 

bioassay fish described by Hunn et al. (1968) and were 

acclimated to test conditions for 24 h before each test. 

Mortalities were recorded at 1, 3, 6, 24, 48, 72, and 96 h.

Commercial grade chloramine-T (lot 12605), obtained 

from Badger Pharmacal, Inc. (Jackson, Wisconsin), was 

used for all tests. The certificate of analysis supplied by 

the company listed the assay as 100.03% available 

chlorine. However, the material yielded 98.4% available 

chlorine when we analyzed it according to the method provided by the manufacturer (U.S. Pharmacopeial Convention, Inc. 1979). All chloramine-T concentrations are 

expressed in milligrams per liter. Concentrations in 

selected test containers were checked analytically at 0, 

6, 24, 48, 72, and 96 h.

Fathead minnows averaging 1.11 g were used to assess 

the effects of three loading rates of fish on the toxicity 

of chloramine-T. We used either 7, 14, or 28 fish to provide loading rates of 0.52, 1.04, or 2.07 g/L. Each set 

was exposed to a series of concentrations of the test 

chemical.

We tested the effects of the presence of fish food on 

the toxicity of chloramine-T by using concentrations of 

xl (0.07 g/L), x2, and x3 the recommended feeding 

rate for fish at 12° C about 5 % of their body weight per 

day (Piper et al. 1982). On the basis of the weight of the 

fish, 0.07, 0.13, or 0.20 g/L of Nelsons' Sterling Silver 

Cup No. 1 fish food was added to each test vessel. The 

feed contained crude protein 48%, crude fat 14%, crude 

fiber 3.6%, and ash 12.0%. Chloramine-T was administered immediately after the feed was added.

The effects of fecal matter on the toxicity of 

chloramine-T were determined by adding three concentrations of a slurry of feces and bottom materials (referred 

to here as fecal material) collected from a fish production raceway. To determine the amount of fecal material 

present in the slurry, we drained the excess water and 

dried five 5-mL portions of the material in an oven at 

100° C for 24 h; the mean dry weight per portion was

0.08 g/mL. We then added portions of the wet fecal 

material to test vessels to yield dry weight equivalent 

concentrations of 0.017, 0.033, and 0.067 g/L of the 

material. After adding the material, we manually stirred 

the contents of each jar, added the fish, and then the 

chloramine-T at concentrations of 2 to 80 mg/L.

Concentrations of chloramine-T in the test waters were 

determined by high performance liquid chromatography 

(HPLC), according to methods described by Bills et al. 

(1988).

Statistical Analysis

The methods of Litchfield and Wilcoxon (1949) were 

used to compute the LC50's (concentration calculated to 

produce 50% mortality) and 95% confidence intervals.

Results and Discussion

Fish Loading Rates

The toxicity of chloramine-T increased as the fish 

loading rate increased from 0.52 to 2.07 g/L; 24-h LC50's 

were 36.0 and 14.0 mg/L, respectively (Table 1). However, the 96-h LC50's were not significantly different 

among the three loading rates. The higher loading rates 

may have stressed the fish and increased the toxicity, even 

though oxygen concentrations were maintained above 

stressful levels (>50% saturation) for fathead minnows. 

Concentrations of chloramine-T decreased over timeespecially at the higher fish loading rates (Table 2). The 

additional fish apparently absorbed more of the chemical 

from solution. The decreased chloramine-T concentrations 

may have offset the increased toxicity associated with 

crowding. By 96 h, there were no significant differences 

in toxicity among the different loading rates.

Table 1. Toxicity of chloramine-T to fathead minnows at 

three different loading rates offish in soft water at 12 ° C.

Fish loading 

rate

(g/L)

0.52

1.04

2.07

LC50 (mg/L) 

95% confidence

24 h

36.0

31.3-41.3

22.5

19.6-25.8

14.0

10.0-19.4

and 

interval

96 h

8.50

7.24-9.98

7.00

6.52-7.51

8.00

7.49-8.54
Table 2. Concentrations (mg/L) of chloramine-T remaining after treatments with the chemical in the presence of 

fish, fish feed, fecal material, fish and fish feed, and fish and fecal material in soft water at 12° C.

Time 

00

0

6

24

48

72

96

0

6

24

48

72

96

0

6

24

48

72

96

Treatment 

concention 

(mg/L)

6

6

6

6

6

6

10

10

10

10

10

10

40

40

40

40

40

40

0.52

5.92

5.92

5.64

5.40

4.91

4.54

9.96

9.87

9.33

8.79

8.01

7.74

39.84

39.57

36.97

Fish 

(g/L)

1.04

5.93

6.54

5.34

4.94

4.24

3.93

10.05

9.89

9.03

7.92

_b

38.94

38.55

_b

Fish feed Fecal material Fish and fish feed 

(g/L) (g/L) (g/L)

Refer2.07 0.07 0.13 0.20 0.02 0.03 0.07 encea

5.74 4.45 3.37 2.28 5.01 4.89 4.75 5.73

5.96 4.14 3.23 2.18 5.14 4.81 4.78 5.66

5.10 3.57 2.46 1.38 5.16 4.68 4.60 5.44

4.33 2.88 1.51 0 5.05 3.91 4.08 5.05

3.56 2.17 0.96 0 4.86 3.29 3.55 4.70

2.94 1.44 1.02 0 4.51 2.81 3.12 4.08

9.70 7.66 5.80 4.53 8.43 8.14 8.09 9.67

9.35 7.51 5.52 4.29 8.56 8.40 8.02 9.58

8.10 6.62 4.24 3.04 8.67 8.34 7.81 9.08

6.54 5.56 2.92 1.71 8.37 7.87 7.14 8.21

5.24 4.85 2.03 0.96 8.19 7.47 6.45 7.54

5.61 4.05 1.26 0 7.77 7.02 5.87 -

38.57 34.37 30.99 26.42 34.17 34.68 32.31 39.07

37.50 34.68 30.01 25.34 34.64 34.15 32.06 38.59

- 32.96 27.98 23.48 35.81 35.83 33.17 -

- 31.45 25.14 20.37 35.04 34.98 32.13 -

- 30.55 24.09 18.17 35.07 34.34 31.43 -

- 29.27 21.94 15.13 34.57 33.72 30.35 -

0.07

4.46

4.31

3.43

2.38

1.64

0.91

7.98

7.55

6.14

4.63

3.57

2.53

34.08

32.27

0.13

3.28

3.46

2.08

1.04

0

0

6.08

5.83

3.98

2.38

1.38

0.60

28.31

27.09

0.20

2.30

2.27

1.12

0

0

0

4.52

4.18

2.55

1.17

0

0

25.61

23.68

Fish and 

fecal material 

(g/L)

Reference3

4.99

5.15

5.22

4.78

4.19

3.57

8.64

8.57

8.59

7.76

6.86

6.19

35.38

34.90

_

0.02

5.29

5.09

4.99

4.40

3.91

3.35

8.95

8.44

8.29

7.43

6.76

5.55

35.26

34.24

0.03

5.15

4.83

4.64

3.98

3.39

2.66

8.68

8.18

7.83

6.86

6.28

5.55

34.25

32.91

0.07

5.04

4.68

4.41

3.65

3.03

2.38

8.48

7.96

7.41

6.31

5.53

4.74

34.11

32.37

aReference solution contained only water and chloramine-T. 

bAll fish died.

Fish Feed

Test solutions with added fish feed were less toxic than 

those without feed especially after 96 h of exposure 

(Table 3): The 96-h LCSO's were 7.30 mg/L for solutions without feed, but 20.8 mg/L for solutions containing 0.20 g/L of feed. The presence of fish feed resulted 

in a loss of chloramine-T. At the 6 mg/L concentration, the addition of 0.07 g/L of fish feed decreased 

chloramine-T concentrations 80% within 96 h and 

0.20 g/L decreased the concentrations to below the detection limit within 48 h. At the 10 mg/L concentration of 

chloramine-T, the loss was significantly higher when 0.13 

or 0.20 g/L of feed was added than when 0.07 g/L was 

added (Table 2). We believe the loss of chloramine-T from 

solution was due to adsorption on feed particles as well 

as to depletion of chlorine due to the oxidation of the 

organic material.

Table 3. Toxicity of chloramine-T to fathead minnows in 

soft water containing four different amounts of fish 

feed.

Amount 

of feed 

(g/L)

0

0.07

0.13

0.20

95

24 h

32.3

25.5-40.

28.5

24.5-33

28.4

24.4-33,

28.0

24.1-32,

LC50 (mg/L) and 

% confidence interval

96 h

7.30

.9 6.71-7.94

14.2

.1 12.5-16.1

17.2

.0 14.4-20.4

20.8

.6 17.9-24.1
Fecal Material

The presence of fecal material decreased the toxicity 

of the chloramine-T solutions in 96-h exposures (Table 4). 

The 96-h LCSO's for the solutions containing the three 

levels of fecal material ranged from 12.0 to 17.4 mg/L, 

in comparison with 7.40 mg/L for solutions that contained 

no fecal material. However, there were no significant differences among the LCSO's at the 24-h exposures. The 

presence of fecal material decreased the chloramine-T 

concentrations at all three loading levels. As fecal material 

levels increased, the loss of chloramine-T also increased. 

The decrease in chloramine-T concentration was independent of the initial concentrations and was directly correlated with the amount of fecal material added (Table 2).

Fish Plus Fish Feed

The presence of fish in combination with feed caused 

the largest loss of chloramine-T observed in any of the 

combinations of the materials tested (Table 2). The loss 

of chloramine-T was 100% over 72 h at 6 mg/L with 

0.13 g/L feed plus fish, and 100% over 48 h at 0.20 g/L. 

Reductions were most rapid between 0 and 24 h. The 

higher the level of feed added, the more rapid the 

reduction in chloramine-T concentration. Therefore, 

chloramine-T would be more effective if the fish were 

not fed before treatment.

Fish Plus Fecal Material

In most tests, the presence of fish in combination with 

fecal material decreased chloramine-T concentrations to 

levels similar to those caused by fish plus feed, although 

the reductions were less significant (Table 2). In 96 h, 

the 6 mg/L concentration of chloramine-T was decreased 

by about 67% and the 10 mg/L concentration was reduced 

by about 50%. In general, fish culturists must be aware 

that treatments with chloramine-T may not be effective 

if the system contains high levels of oxidizable material 

or material that can absorb the chemical and reduce its 

effective concentration.

References

ASTM Committee E-35 on Pesticides. 1980. Standard practice 

for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians, E729-80. Pages 1-25 in Annual 

book of ASTM standards, Part 46. End use and consumer

Table 4. Toxicity of chloramine-T to fathead minnows in 

soft water containing four different amounts of fish 

fecal material.

Amount 

of fecal 

material 

g/15L

0

0.02

0.03

0.07

LC50 (mg/L) 

95% confidence

24 h

26.5

20.7-33.9

25.5

21.1-30.8

24.5

20.4-29.4

25.5

21.1-30.8

and 

interval

96 h

7.40

6.65-8.23

12.0

10.3-13.9

12.2

10.8-13.8

17.4

14.6-20.7

products. American Society for Testing and Materials, Philadelphia, Pa.

Bills, T. D., L. L. Marking, V. K. Dawson, and J. J. Rach. 

1988. Effects of environmental factors on toxicity of 

chloramine-T to fish. U.S. Fish Wildl. Serv., Invest. Fish 

Control. 96. 6 pp.

Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. Environ. Prot. Agency, 

Ecol. Res. Serv., EPA-660/3-75-009. 61 pp.

Cross, D. G., and P. A. Hursey. 1973. Chloramine-T for the 

control of Ichthyopthirius multifilis (Fouquet). J. Fish Biol. 

5:789-798.

From, J. 1980. Chloramine-T for control of bacterial gill disease. 

Prog. Fish-Cult. 42:85-86.

Hunn, J. B., R. A. Schoettger, and E. W. Whealdon. 1968. 

Observations on handling and maintenance of bioassay fish. 

Prog. Fish-Cult. 30:164-167.

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

method of evaluating dose-effect experiments. J. Pharmacol. 

Exp. Ther. 96:99-113.

Piper, R. G., I. B. McElwain, L. E. Orme, J. P. McCraren, 

L. G. Fowler, and J. R. Leonard. 1982. Fish hatchery 

management. U.S. Fish Wildl. Serv., Washington, D.C. 

517 pp.

Snieszko, S. F. 1981. Bacterial gill disease of freshwater fishes. 

U.S. Fish Wildl. Serv., Fish Dis. Leafl. 62. 11 pp.

U.S. Department of Agriculture. 1986. Interregional research 

project no. 4. Guidelines for IR-4 investigations. New Jersey 

Agricultural Experiment Station, Cook College, Rutgers 

University, New Brunswick, N.J. 6 pp.

U.S. Pharmacopeial Convention, Inc. 1979. Chloramine-T 

assay. Pages 1057-1058 in U.S. Pharmacopia. 20th Revision. 

Mack Publishing Co., Easton, Pa.

Van Duijn, C. 1967. Diseases of fishes, 2nd ed. Charles C. 

Thomas, Publisher, Springfield, 111. 309 pp.
Bills, Terry D., Leif L. Marking, Verdel K. Dawson, and George E. Howe. 

1988. Effects of organic matter and loading rates of fish on the toxicity 

of chloramine-T. U.S. Fish Wildl. Serv., Invest. Fish Control 97. 4 pp.

The present study was designed to determine the effects of different fish loading 

rates, feed levels, and residual fecal material on availability and toxicity of 

chloramine-T (n-sodium-n-chloro-para-toluenesulfonamide), a chemical used for 

the control of bacterial gill disease. Toxicity of chloramine-T increased as loading 

rates of fathead minnows (Pimephalespromelas) increased from 0.52 to 2.07 g/L 

of water in 24-h exposures. The 96-h LCSO's were 7.30 mg/L for solutions 

without feed and 14.2 to 20.8 mg/L after the addition of 0.07 to 0.20 g/L of 

feed. The concentration of chloramine-T in water decreased more rapidly in the 

presence of fish than in the presence of only feed or fecal material. The losses 

of chloramine-T were greatest in the presence of combinations of fish and feed 

or fish and fecal material.

Key words: Toxicity, chloramine-T, fish, inactivation, organic matter.

Bills, Terry D., Leif L. Marking, Verdel 

1988. Effects of organic matter and l

of chloramine-T. U.S. Fish Wildl. Se

The present study was designed to determ

rates, feed levels, and residual fecal ma

chloramine-T (n-sodium-«-chloro-para-tolu

the control of bacterial gill disease. Toxicity 

rates of fathead minnows (Pimephales prom

of water in 24-h exposures. The 96-h LC

without feed and 14.2 to 20.8 mg/L after 

feed. The concentration of chloramine-T in 

presence of fish than in the presence of on

of chloramine-T were greatest in the prese

or fish and fecal material.

Key words: Toxicity, chloramine-T, fish

Bills, Terry D., Leif L. Marking, Verdel K. Dawson, and George E. Howe. 1988. Effects of organic 

matter and loading rates offish on the toxicity of chloramine-T. U.S. Fish Wildl. Serv., Invest. Fish 

Control 97. 4 pp.

The present study was designed to determine the effects of different fish loading rates, feed levels, 

and residual fecal material on availability and toxicity of chloramine-T (n-sodium-n-chloro-paratoluenesulfonamide), a chemical used for the control of bacterial gill disease. Toxicity of chloramine-T 

increased as loading rates of fathead minnows (Pimephalespromelas) increased from 0.52 to 2.07 g/L 

of water in 24-h exposures. The 96-h LC50's were 7.30 mg/L for solutions without feed and 14.2 to 

20.8 mg/L after the addition of 0.07 to 0.20 g/L of feed. The concentration of chloramine-T in water 

decreased more rapidly in the presence of fish than in the presence of only feed or fecal material. 

The losses of chloramine-T were greatest iri the presence of combinations of fish and feed or fish and 

fecal material.

Key words: Toxicity, chloramine-T, fish, inactivation, organic matter.

U.S. Government Printing Office: 1989 673-208/00001

Bills, Terry D., Leif L. Marking, Verdel K. Dawson, 

matter and loading rates of fish on the toxicity of ch

Control 97. 4 pp.

The present study was designed to determine the eff

and residual fecal material on availability and toxic

toluenesulfonamide), a chemical used for the control o

increased as loading rates of fathead minnows (Pimeph

of water in 24-h exposures. The 96-h LCSO's were 7.

20.8 mg/L after the addition of 0.07 to 0.20 g/L of fe

decreased more rapidly in the presence of fish than 

The losses of chloramine-T were greatest in the presen

fecal material.

Key words: Toxicity, chloramine-T, fish, inactivatio

(Reports 87 through 89 are in one cover.)

87. Ethyl-/?-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.

(Reports 90 and 91 are in one cover.)

90. Accumulation and Loss of 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) by Fish: Laboratory Studies, by V. K. Dawson, 

J. B. Sills, and Charles W. Luhning. 1982. 5 pp.

91. Effects of Synergized Rotenone on Nontarget Organisms in Ponds, by R. M. Burress. 1982. 7 pp.

(Reports 92 through 94 are in one cover.)

92. Acute and Chronic Toxicity of Rotenone to Daphnia magna, by J. J. Rach, T. D. Bills, and L. L. Marking. 1988. 5 pp.

93. Toxicity of Rotenone to Developing Rainbow Trout, by T. D. Bills, J. J. Rach, and L. L. Marking. 1988. 3 pp.

94. Oral Toxicity of Rotenone to Mammals, by L. L. Marking. 1988. 5 pp.

95. Deposition and Persistence of Rotenone in Shallow Ponds During Cold and Warm Seasons, by P. A. Gilderhus, V. K. 

Dawson, and J. L. Alien. 1988. 7 pp.

NOTE: Use of trade names does not imply U.S. Government endorsement of commercial products.
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