5H , — ye || i Fi Sh s INVESTIGATIONS IN FISH CONTROL ES 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 . es CE? Pee COS SRT { . ee fe Ope eel rst .) a BS i © 5 & A ws 2 .Ubsaty a a ? ees u " IN OE OQ SE BREE phy tS B) lL oeS 4 ss oN 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 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. Chloramine- T— Environmental aspects. I. Bills, T. D. II. Effects of organic matter and loading rates of fish 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 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 condi- tions. The toxicity (96-h LC50) was 2.80 mg/L for rainbow trout, Salmo gairdneri; 3.75 mg/L for channel catfish, Jctalurus 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 tox- icity 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 recom- mended 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 Chloramine-T (n-sodium-n-chloro-para-toluenesulfona- mide) was shown by From (1980) to be effective for con- trolling bacterial gill disease (BGD), which is one of the most common diseases of hatchery-reared salmonids and causes more fish losses than any other bacterial disease. The disease is strongly mediated by the stressful environ- mental conditions and marginal nutrition commonly associated with intensive culture. Flavobacteria and flexi- bacteria are associated agents (Snieszko 1981). Gills damaged by BGD pathogens are also prone to secondary invasion by fungi (Warren 1981). Because BGD is a limit- ing factor in fish production, a control agent, such as chloramine-T, is needed for use on food fish. Approval by the U.S. Food and Drug Administration will be required. Researchers have reported that the activity of chloramine-T is affected by pH, water hardness, and temperature. Tooby et al. (1975) found that the toxicity of chloramine-T to fish and eggs increased as pH and water hardness decreased. Cross and Hursey (1973) observed that chloramine-T was more toxic to fish in soft, acid waters than in hard, alkaline waters, and less toxic at 10° C than at high temperatures. The purpose of the present study was to delineate the five factors that affect the toxicity of chloramine-T to selected species of fish: (1) sensitivity differences between selected coldwater and warmwater fish species; (2) ef- fects of pH, water hardness, and temperature; (3) per- sistence of toxicity in solutions over a 4-week period; (4) toxicity and observed effects at recommended use pattern concentrations for rainbow trout (Salmo gaird- neri); and (5) the time-independent toxicity. i) 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. Recon- stituted test waters were prepared according to standard- ized 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 main- tain the selected pH (+0.2 unit). Temperatures were reg- ulated 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 bicar- bonate using the procedure of Marking (1975). Rainbow trout, fathead minnows (Pimephales pro- melas), and channel catfish (Ictalurus punctatus), ob- tained from a Federal fish hatchery or produced at the National Fisheries Research Center, LaCrosse, Wiscon- sin, were maintained according to the standard proce- dures 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 tempera- ture, 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 of fish 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. Pharmacopeial 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 um 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 per- formed 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: sta- tionary 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; wave- length, 229 nm; and attenuation, 0.10 absorption units. A 0.45-um disposal filter assembly was used to filter the sample. Reagents were acetonitrile, HPLC grade; water, HPLC grade; phosphoric acid, American Chemical Society re- agent grade 0.2 M, 13.6 mL diluted to 1 L with HPLC water; monobasic potassium phosphate, American Chem- ical Society reagent grade 0.2 M, consisting of 27.2 g of KHjPO, 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 KH>PO, 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 ana- lytically each week and the toxicity was compared with that of fresh solutions. Deactivation indices were calcu- lated 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 inter- vals. 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 LCS50’s (mg/L) were 2.80 for rainbow trout, 3.75 for channel catfish, and 7.30 for fathead min- nows (Table 1). Rainbow trout (coldwater) and channel catfish (warmwater) were thus twice as sensitive as fat- head minnows (warmwater). Table 1. Toxicity of chloramine-T to three species of fish in soft water at 12°C. 96-h LC50 and 95% Mean weight confidence interval Species Lot Source? (g) (mg/L) Rainbow trout 8640 Erwin NFH 0.73 2.80 2.41-3.26 Fathead minnow 8631 LaCrosse NFRC 1.11 7.30 6.71-7.94 Channel catfish 8632 LaCrosse NFRC 1.50 3.75 3.30-4.26 4NFH = National Fish Hatchery; NFRC = National Fisheries Research Center. Influence of Temperature, Water Hardness, and pH The toxicity of chloramine-T was affected by water characteristics, and alterations in toxicity were consistent between the two species. The chemical was more toxic in warm water than in cold water in exposures of 24 h or less. For example, the 3-h LC50 for rainbow trout was 28.0 mg/L at 17° C and 43 mg/L at 12° C; at 7° C there was no mortality at 60 mg/L (Table 2). Channel catfish did not respond as quickly to the chemical; there was no mortality in 3 h at 60 mg/L at any temperature (Table 3). After 6 h at temperatures of 22, 17, and 12° C, the LC50’s were 14.0, 25.3, and >60.0 mg/L, respectively. Tem- perature apparently affected the time of response rather than the ultimate toxicity of chloramine-T; the 96-h LC50’s did not differ significantly for either species. Water hardness had only a slight effect on the tox- icity of chloramine-T to rainbow trout and no effect on channel catfish (Tables 2 and 3). For rainbow trout, the 96-h LCS50 was 7.35 mg/L in very soft water (10-13 mg/L as CaCO3) 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 Soft WES >60.0 >60.0 28.0 18.0 11.7 4.30 22.8-34.4 15.3-21.1 10.5-13.0 3.69-5.01 12 Soft Ved >60.0 43.0 17.5 14.0 6.90 2.80 36.9-50.1 14.8-20.7 11.3-17.3 6.31-7.54 2.41-3.26 17 Soft ES >60.0 28.0 10.0 8.00 4.90 2.80 22.7-34.5 9.07-11.0 7.57-8.46 4.48-5.35 2.27-3.46 12 Very soft 8.1 >60.0 >60.0 >60.0 38.7 20.2 7e35 33.9-44 .2 17.7-23.0 6.92-7.80 12 Soft 8.1 >60.0 >60.0 >60.0 47.5 25.4 9.00 - — 21.0-30.6 9.33-9.72 12 Hard 8.1 >60.0 >60.0 >60.0 45.0 25.0 10.0 38 .5-52.6 20.7-30.1 9.04-11.1 12 Very hard 8.1 >60.0 >60.0 >60.0 49.0 25.5 14.2 —- — 21.2-30.7 12.2-16.5 12 Soft 6.5 55.8 13.5 8.22 6.05 2.81 1.89 48.4-64.3 11.2-16.2 7.71-8.76 5.17-7.08 2.41-3.27 1.63-2.19 12 Soft 8.5 >60.0 >60.0 >60.0 55.7 46.0 11.0 49.1-63.1 39.5-53.6 9.78-12.4 12 Soft 9.5 >60.0 >60.0 >60.0 >60.0 >60.0 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. Duration of test (h) Temperature (°C) Hardness pH 1 3 6 12 24 96 12 Soft U5 >60.0 >60.0 >60.0 14.2 10.0 3.75 12.2-16.5 9.07-11.0 3.30-4.26 17 Soft ES >60.0 >60.0 25.3 14.2 7.20 3.73 21.0-30.4 12.2-16.5 6.78-7.64 3.29-4.22 22 Soft US >60.0 >60.0 14.0 9.00 5.63 3.80 12.0-16.3 8.51-9.51 5.08-6.24 3.33-4.34 12 Very soft 8.1 >60.0 >60.0 >60.0 >60.0 28.0 7.70 24.1-32.6 6.98-8.49 12 Soft 8.1 >60.0 >60.0 >60.0 >60.0 30.5 11.0 26.3-35.4 9.13-13.2 12 Hard 8.1 >60.0 >60.0 >60.0 >60.0 33.0 7.80 29.2-37.3 6.53-9.31 12 Very hard 8.1 >60.0 >60.0 >60.0 >60.0 37.0 9.80 32.7-41.8 8.64-11.1 12 Soft 6.5 >60.0 27.0 10.0 5.60 2.85 1.75 21.0-35.0 9.07-11.0 5.21-6.01 2.52-3.22 1.48-2.06 12 Soft 8.5 >60.0 >60.0 >60.0 >60.0 51.5 10.5 46.8-56.7 9.14-12.1 12 Soft 9.5 >60.0 >60.0 >60.0 >60.0 >60.0 12.3 9.75-15.5 water (280-320 mg/L as CaCO3). Tooby et al. (1975) Persistence 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) com- pared 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 1hto x1, <3, and X5 the recommended use pattern concentration (12 mg/L) did not differ from those of con- trol 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 mor- tality with overtreatment. The toxicity of aged solutions of chloramine-T de- creased 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. 96-h LCS0 (mg/L) Aging period and 95% confidence Deactivation (weeks) interval index? 0 2.85 1.0 2.57-3.16 1 4.10 1.44 3.54-4.72 2 3.80 1.33 3.34-4.32 3 8.00 2.81 6.15-10.4 4 6.00 2.10 5.27-6.82 41C50 of aged solution LCS0 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 time (weeks) 6 0 @ -6. 10 a 0 6.12 10.26 40.59 5.84 9.88 39.33 1 5.82 9.82 39.60 5.44 9.37 37.46 2 5.43 9.34 37.48 5.62 9.38 39.22 3 5.41 9.33 38.98 5.58 9.66 41.11 4 5.50 9.56 40.81 LCS0 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 ex- ample, 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 tox- icity 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 devia- tions (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 LCS0’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 of fish ex- posed to chloramine-T did not continue beyond 96 h; the time-independent LC50’s were similar to the 96-h LC50’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, macroinver- tebrates, 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. Calculated concentration (mg/L) 6 10 40 6 10 40 6 10 40 5.78 9.58 37.94 5.88 9.87 39.60 5.94 SK 973 DWH D8 G6) S278) 5.89 9.87 40.29 10.02 39.65 Committee on Methods for Toxicity Tests with Aquatic Organ- isms. 1975. Methods for acute toxicity tests with fish, macro- invertebrates, 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. LCS0’s (mg/L) and 95% confidence intervals Fish source and type of test 96 h TILC50? Lot 8675 Static 4.60 — 4.14-5.11 —- = Flow-through 30.0 24.3 27.6-32.6 21.5-27.5 Lot 8706 Static 7.00 — 6.44-7.61 = = Flow-through 23.0 23.0 20.2-26.2 20.1-26.2 *Time-independent LC50. Marking, L. L. 1972. Methods of estimating the half-life of biological activity of toxic chemicals in water. U.S. Fish Wildl. Seryv., Invest. Fish Control 46. 9 pp. Marking, L. L. 1975. Toxicological protocol for the develop- ment of piscicides. Pages 26-31 in P. H. Eschmeyer, ed. Rehabilitation of fish populations with toxicants: A sym- posium. 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. 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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 (n-sodium-n-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 de- creased the toxicity of the chemical. The 96-h LCSO 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 (n-sodium-n-chloro-para-toluenesulfo- namide) 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 respon- sible for BGD infections, all known agents are gram- negative bacteria—myxobacteria, aeromonads, and pseudomonads (Snieszko 1981). The disease is highly con- tagious 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 inacti- vated 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 com- pound was more toxic in acid than in alkaline water. The purpose of the present study was to determine (1) the effects of fish 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 CaCO3) 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, Wiscon- sin, were used as the test organisms. They were main- tained 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 96h. 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 pro- vided by the manufacturer (U.S. Pharmacopeial Conven- tion, 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 pro- vide 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 x1 (0.07 g/L), <2, 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 admin- istered immediately after the feed was added. The effects of fecal matter on the toxicity of chloramine-T were determined by adding three concen- trations of a slurry of feces and bottom materials (referred to here as fecal material) collected from a fish produc- tion 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). How- ever, 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 time— especially 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 of fish in soft water at 12° C. LCS5O0 (mg/L) and Fish loading 95% confidence interval rate (g/L) 24h 96 h 0.52 36.0 8.50 31.3-41.3 7.24-9.98 1.04 22.5 7.00 19.6-25.8 6.52-7.51 2.07 14.0 8.00 10.0-19.4 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. Fish and Treat- Fish Fish feed Fecal material Fish and fish feed fecal material ee (g/L) (g/L) (g/L) (g/L) (g/L) OTIC E 1) yoann cA I erence NE ak Al a a Time tion Refer- Refer- (h) (mg/L) 0.52 1.04 2.07 0.07 0.13 0.20 0.02 0.03 0.07 ence* 0.07 0.13 0.20 ence* 0.02 0.03 0.07 0 6 5.92 5.93 5.74 4.45 3.37 2.28 5.01 4.89 4.75 5.73 4.46 3.28 2.30 4.99 5.29 5.15 5.04 6 6 5.92 6.54 5.96 4.14 3.23 2.18 5.14 4.81 4.78 5.66 4.31 3.46 2.27 5.15 5.09 4.83 4.68 24 6 5.64 5.34 5.10 3.57 2.46 1.38 5.16 4.68 4.60 5.44 3.43 2.08 1.12 5.22 4.99 4.64 4.41 48 6 5.40 4.94 4.33 2.88 1.51 0 5.05 3.91 4.08 5.05 2.38 1.04 0 4.78 4.40 3.98 3.65 72 6 4.91 4.24 3.56 2.17 0.96 0 4.86 3.29 3.55 4.70 1.64 0 0 4.19 3.91 3.39 3.03 96 6 4.54 3.93 2.94 1.44 1.02 0 4.51 2.81 3.12 4.08 0.91 0 0 SES 3352-00) 2-38 0 10 9.96 10.05 9.70 7.66 5.80 4.53 8.43 8.14 8.09 9.67 7.98 6.08 4.52 8.64 8.95 8.68 8.48 6 10 9.87 9.89 9.35 7.51 5.52 4.29 8.56 8.40 8.02 9.58 7.55 5.83 4.18 8.57 8.44 8.18 7.96 24 10 9.33 9.03 8.10 6.62 4.24 3.04 8.67 8.34 7.81 9.08 6.14 3.98 2.55 8.59 8.29 7.83 7.41 48 10 8.79 7.92 6.54 5.56 2.92 1.71 8.37 7.87 7.14 8.21 4.63 2.38 1.17 7.76 7.43 6.86 6.31 72 10 SOI" 5-247"4785 21030196 8.19 7-47 6.451 7:54 3.57 1.38) 0 6.86 6.76 6.28 5.53 96 10 7.74 — 5.61 4.05 1.26 0 7.77 7.02 5.87 — 2.53 0.60 0 Opie) Seb) sks) 4in74! 0 40 39.84 38.94 38.57 34.37 30.99 26.42 34.17 34.68 32.31 39.07 34.08 28.31 25.61 35.38 35.26 34.25 34.11 6 40 39.57 38.55 37.50 34.68 30.01 25.34 34.64 34.15 32.06 38.59 32.27 27.09 23.68 34.90 34.24 32.91 32.37 24 40 36.97 —> — 32.96 27.98 23.48 35.81 35.83 33.17 — — — = — — — 48 40 = = — 31.45 25.14 20.37 35.04 34.98 32.13 — — —- — — - =—- = 72 40 —- = — 30.55 24.09 18.17 35.07 34.34 31.43 — _ _- — — — - — 96 40 = = — 29.27 21.94 15.13 34.57 33.72 30.35 — — - — — - -—- = Reference solution contained only water and chloramine-T. Pall fish died. Fish Feed Table 3. Toxicity of chloramine-T to fathead minnows in soft water containing four different amounts of fish Test solutions with added fish feed were less toxic than feed. those without feed—especially after 96 h of exposure (Table 3): The 96-h LC50’s were 7.30 mg/L for solu- y nount 95 Z G0 se eae \ tions without feed, but 20.8 mg/L for solutions contain- oF feeg sath OE ar aay ing 0.20 g/L of feed. The presence of fish feed resulted (g/L) 24h 96h ineawlosssOrechloramine- yAtithe 6ime/, concentra- == tion, the addition of 0.07 g/L of fish feed decreased 0 32.3 7,30 chloramine-T concentrations 80% within 96 h and 25.5-40.9 6.71-7.94 0.20 g/L decreased the concentrations to below the detec- 0.07 28.5 14.2 tion limit within 48 h. At the 10 mg/L concentration of 94.5-33.1 1) Gi 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 0.13 28.4 17,2 added (Table 2). We believe the loss of chloramine-T from 24.4-33.0 14.4-20.4 solution was due to adsorption on feed particles as well as to depletion of chlorine due to the oxidation of the organic material. 0.20 28.0 20.8 24.1-32.6 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 dif- ferences 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 indepen- dent of the initial concentrations and was directly cor- related 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 O 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, macroinverte- brates, 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 LCSO (mg/L) and of fecal 95% confidence interval material g/15 L 24h 96 h 0 26.5 7.40 20.7-33.9 6.65-8.23 0.02 D3 5 12.0 21.1-30.8 10.3-13.9 0.03 24.5 12.2 20.4-29.4 10.8-13.8 0.07 25.5 17.4 21.1-30.8 14.6-20.7 products. American Society for Testing and Materials, Phila- delphia, 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 Organ- isms. 1975. Methods for acute toxicity tests with fish, macro- invertebrates, 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, Ill. 309 pp. “TayeU TURSIO ‘UOTBATORUT “Ys ‘L-ouTUeIOTYS ‘AIOIXO] :spiom Aay ‘[ellayeul [ej pue YSlj 10 pody pue YSTJ JO SUOIBUIQUIOS Jo sdUaSoId dU} UT Jso}V91B JIOM ]-IUTWILIOTYS JO Sasso] eyL, ‘Jeliaeul fede} JO psay ATUO JO aduUaso1d oy} UT UY} YsIJ Jo douasaid ay) UT A[pIdes1 a1oW! paseasoap 19}@M UT J-IUTWIRIOTYS JO UONLIJUIOUOD OYJ, Pee} JO T/3 YZ'O O} LO'O JO UOTIIppe oy} Joye T/3w g°OZ 0} Zp] Pue psaj NOY SUONNIOS OJ T/3W YE'L 919M S,QSOT 4-96 SUL ‘Soinsodxe y-pZ Ur J9}eM Jo 1/3 LOZ 01 ZS ‘0 Wor posvaroUr (spjawmioud sappydauig) SMOUUTUW peayyey JO $a}eI SuIpeo] se paseasour L-suTUTeIOTYS JO AWOIXO], ‘oseasip |[I3 [eLI}Deq JO [O.UOD OY} JO} pasn [eoruIayo ke ‘(aprureUOJ[NsauaNIo} -eied-O1O[YS-u-WINIpOs-u) J-oUTUeIO[YD Jo AjOIxo} pue AYI[IQeyIeAe uO [eIIa\eUI [eooy [eNpIsol pue “S]QAQ] Posy ‘Saye1 SUIPLO] YSTJ JUOIOJJIP JO S}DaJJo OY) OUTWID}aP 0} pousIsap sem Apnys yuasaid oy ‘dd p "16 josuog YSLY “ISIAUT *AIOS “PITA UST “S') * L-eUTUTeO[YD Jo AjIDTx0} BY} UO Ys Jo Saje.i1 SuIpeo] pue 19}jeuI HULSIO JO $DIJJA “8861 “OMOH “| 9310aH pue ‘uosmed “y [epse,a ‘Bune “Ts97T ‘'q AueL ‘sig “JOWCU OTULSIO ‘UONATORUT ‘YsIy ‘L-oUTWIeIOTYO ‘AWOIXO] :spaom Kay "[eLIayeU [edo pue YSIf 10 poo} puke Yslj JO SUOBUTQUIOD Jo sdUaSoI1d OY) UT 3S}BOIT OIOM J-OUTUIPIO[YS JO SOSSO] BY], “[PIIaeUI [ed9J 10 paay ATUO Jo dduasoid ay) UI UY) YSIJ Jo souasaI1d oy} ul Aypides o10W! paseedap 1a}eM Ul J-SUTUIOTYO Jo UOTJeIJUZOUOD dy], ‘p2oy JO T/3 070 1 L0'0 JO UONIppe oy) Jaye T/3W g°0Z 0) Zp] Pu poo inoyIM SUOTINJOS JOJ T/B3UI YE*L 219M S,OSDT Y-96 BU ‘Somnsodxa y-p7 UI J9}eM Jo 1/3 LOZ 0} ZS ‘0 Woy pesearoUt (spjawoud sajpydaulig) SMOUUTW PerYIe] JO soyeI SUIPLO| SB PasedIOUT J -SUTUTRIOTY Jo AWOIKO], “aseasIp [[I3 [eLI9}9eq Jo [oNUOD aU) JO} pesn Jeoruayo e *(eprueuosjnsousno}-e1ed-oJoyYyo-u-WINIpos-u/) [.-uTUeIOTYO Jo Ayorxo} pue AjyIqE[eae uO JeLJayeU [eooy [eNpIsol pue ‘s[aaa] poo} ‘soyeI BUIPLO] YSLJ JUDIAJJIP JO s}oaJyo oy} OUTWLIOJap 0} pousIsap sem Apmys juasaid uy, ‘dd p “16 Jo4uoD ysi] “ISaAuy ‘AIS “PIA USI “SA *L-ouruTes0TYD Jo AJIIIXO} BY} UO YS JO Sayes SuIpeo] pue 19)jJeUl ULSI JO $}IIJIq °8861 “OMOH “Y 28100H pur ‘uosmed “y [eploA ‘BunNpeyy “T Jloy “q Auey ‘sypg L0000/802-€29—6 86} :291JjO Buljuig juaWUseAcd ‘S'f 2% “JOVEUW StuesIO ‘UONBATeUT ‘Ysly ‘f-auTWeIOTYO ‘AJOIXO], :spiom Lay "[eria}eu [ej pue Yslj 10 poay puke YsIJ JO SUOIBUIQUIOD JO JdUAasaId a4} UT JSa}BO13 DIAM J-SUTUILIOTYO JO Sasso] UL “[Bl1ayeul [2O9J 10 paay ATUO Jo souasaid ay) UI URY) YSIJ Jo douasoid ay) UI A[pides o1OW paseosoap JOIVM UT J-IUIWEIOTYS JO UOTJRIUIDUOS BY] “P22J JO T/3 QZ O} LO'O JO UONIppe oy} Joye T/3W g°Qz 0} Zp] Pue psoj JNOYIUM SUONINIOS OJ T/BW YE'L 9M S.OSOT Y-96 SUL “Seinsodxa y-pz ul 19}eM JO 1/3 LOZ 01 ZS ‘0 Wor paseasoUl (svjawourd sajpydawuig) SMOUUTW Peayjey JO SajeI SuIpeo] se paseasoul [-SUIWIeIOTYS JO AYOIXOL ‘aseasip [[IS [e119}9eq JO [OIIUOD ay) JO} pasn [eoTUIaYO e ‘(aprueUOJ[NsouUaNjO} -ered-O10[Y9-u-WuNIpos-uv) J -ouTWIeIO[YD Jo AyOIxo) pue Ajl[Iqeyleae uO [elJa}eW [edaJ [enpIsal pue “S[QA9] Poo] ‘Sae1 SUIPEO] YSIJ JUIIAJJIP JO S}OaJJo OY} QUIULIA}ap 0} paUZIsap sem Apnys juasoid ayy ‘dd p “16 os1u0D YSLY “ISAAUT “ATS “[PTLA UST ‘Sf *L-euTUTerOTYD Jo AjIDTxX0) 3Y} UO YS Jo saje.1 SUTPLOT pue 13jjyeur DUBBIO JO $}I2JJA 8861 “2MOH “Y 29109 pur ‘uosmed “y [apsoA ‘BuNpey, “TJ slaq ‘-q Auay ‘sig “JOYVU STULSIO ‘UOTBATOLUT “YSTJ ‘L-oUTWeIOTYO ‘AWIOIxO] :spiom Avy ‘[elIoyeu [eoo} pue YsI 10 posj pue YSIJ JO SUOTBUIQUIOD Jo sdUaSoId OY) UT Jsa}BOIT JOM J-UTUILIOTYO JO SASSO] OY “[PlI9}eUI [ed9J 10 paaj A[UO Jo doUaSeId oY} UI UeY) YsIJ Jo soUasaId 94) ul A[pides d1OU paseoioap 19}eM UI J-UTWIEIOTYS JO UOTJBIUDOUOD OY], “Pooy JO 1/3 0Z'0 © L0'0 JO UONIppe oy) Joye T/3W B07 0} Zp] Pu pad} InoypIM SUOIIN[OS IOJ J/SW YE'L 9M S,QSOT 4-96 OYL ‘Soinsodxa y-pz UI Jo}eM Jo 1/3 LOZ 01 TSO Woy paseaioUt (syjawiosd sajvydaulid) SMOUUTU peasyjey Jo saye1 BUIPRO] SV POSBdIOUT [.-OUTUeIOTYO JO AJOIXO], “aseasIp [[I3 [eLIA}0eq Jo [oIUOD oy) JO} posn [eoTWsY v ‘(eprueUOJ[NsouoN]o}-e1ed-o1O[YO-u-WINIPOs-u) J-sUTUTeIOTYO Jo Ajlorxoy pue Ajypiqepreae uo [eLayeul [eda} [eNplsol pue ‘sfoao, pasj ‘soye1 SUIPROT USI} JUSIOJJIP JO $}D9JJo ay} OUTULIDJOp 0} pousIsop sem Apnys juasaid oy], ‘dd p “16 Jouuod ysiq ‘Isaauy “AIO “[PILAA USTA “S'N. *L-euTWer0[Y9 Jo A}IIXO} JY} UO YSIJ JO SjIV1 SUIPLO] PUL 19}}BUT JULIO JO S}IATIW “SRI “OMOH “| 93100H pue ‘uosmeq “yf [eps9A ‘“SuNpeY “TJ JOT “qd AuoL ‘sytg a es (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. (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. Allen. 1988. 7 pp. NOTE: Use of trade names does not imply U.S. Government endorsement of commercial products. WMC 3 9088 01017 4795 TAKE PRIDE in Amenica eee U.S. DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE 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.