Efficacy and Toxicity of Chloramine T Disinfection on Cisco Fry G. E. MACKEY, M. A. CHALUPNICKI, J. H. JOHNSON USGS Great Lakes Science Center, Tunison Laboratory of Aquatic Science Cortland, NY N. H. RINGLER SUNY College of Environmental Science and Forestry Syracuse, NY D. D. IWANOWICZ USGS National Fish Health Laboratory Kearneysville, WV G. E. MACKEY, M. A. CHALUPNICKI, J. H. JOHNSON USGS Great Lakes Science Center, Tunison Laboratory of Aquatic Science Cortland, NY N. H. RINGLER SUNY College of Environmental Science and Forestry Syracuse, NY D. D. IWANOWICZ USGS National Fish Health Laboratory Kearneysville, WV ABSTRACT: Restoring native cisco (Coregonus artedi) populations into Lake Ontario is a joint effort by the New York State Department of Environmental Conservation, and the U.S. Geological Survey. The restoration of cisco has given rise to new husbandry techniques; in particular protocols to control bacterial pathogens on hatchery raised fish. We evaluated the toxicity of Chloramine T to cisco fry, and its effectiveness as a disinfectant of bacterial flora on the surface of the gills. Ciscoes were exposed to three consecutive daily one hour treatments at concentrations of 0, 5, 10, 20, 40, 80, or160mg/L. Survival of fry tended to be similar at concentrations up to 80mg/L and significantly reduced at concentrations of 160mg/L. We calculated the concentrations of Chloramine T that killed 50% of the cisco fry (LC 50 ) to be 112 mg/L. Following three consecutive daily one hour treatments of 20mg/L Chloramine T, Paracoccus sp. and Acinetobacter lwoffii were the predominant species present on gill surfaces and were significantly greater in abundance following the treatment. The use of Chloramine T as a disinfectant on cisco fry was not effective at controlling gill bacterial presence within concentrations where toxicity was minimal. ABSTRACT: Restoring native cisco (Coregonus artedi) populations into Lake Ontario is a joint effort by the New York State Department of Environmental Conservation, and the U.S. Geological Survey. The restoration of cisco has given rise to new husbandry techniques; in particular protocols to control bacterial pathogens on hatchery raised fish. We evaluated the toxicity of Chloramine T to cisco fry, and its effectiveness as a disinfectant of bacterial flora on the surface of the gills. Ciscoes were exposed to three consecutive daily one hour treatments at concentrations of 0, 5, 10, 20, 40, 80, or160mg/L. Survival of fry tended to be similar at concentrations up to 80mg/L and significantly reduced at concentrations of 160mg/L. We calculated the concentrations of Chloramine T that killed 50% of the cisco fry (LC 50 ) to be 112 mg/L. Following three consecutive daily one hour treatments of 20mg/L Chloramine T, Paracoccus sp. and Acinetobacter lwoffii were the predominant species present on gill surfaces and were significantly greater in abundance following the treatment. The use of Chloramine T as a disinfectant on cisco fry was not effective at controlling gill bacterial presence within concentrations where toxicity was minimal. Gill Bacteria Evaluation An additional subsample of ciscoes were also examined for bacterial colony presence before and after a three consecutive daily one hour 20mg/L Chloramine T treatment. Prior to and following treatment 20 fish were measured, weighed, and their gills were removed for bacterial presence and enumeration. Primary isolation of bacteria was accomplished by placing each set of gills in 1 mL of sterile 0.1 % peptone-0.05 % yeast extract diluent in a sterile 13 x 100 mm tube. We vigorously mixed the gills and buffer solution for seconds. We transferred 20 μL buffer diluent from each set of gills to the surface of R2A agar plates. The agar plates were incubated at 20ºC for 48 to72 h. The resulting individual bacterial colonies were enumerated and the data were presented as the number of colony forming units per gram of gill (CFU/g). Bacterial colonies representing the predominant colony phenotypes were transferred to fresh R2A media and were characterized using standard methods for identification of bacteria. In statistical analyses of our data, we considered P<0.05 to be significant. We determined the LC50 for cisco fry following the three day treatment using the Trimmed Spearman Karber method. Because the Shapiro-Wilks test showed that data were not normally distributed, we used the non-parametric Kruskal-Wallis one-way analyses of variance test to examine differences in survival (response variable) among Chloramine T concentrations (independent variable) as well as the numbers of bacterial colonies present on the gills. When differences were significant, we used Tukey’s pairwise comparison test to determine which concentrations differed significantly in survival. Gill Bacteria Evaluation An additional subsample of ciscoes were also examined for bacterial colony presence before and after a three consecutive daily one hour 20mg/L Chloramine T treatment. Prior to and following treatment 20 fish were measured, weighed, and their gills were removed for bacterial presence and enumeration. Primary isolation of bacteria was accomplished by placing each set of gills in 1 mL of sterile 0.1 % peptone-0.05 % yeast extract diluent in a sterile 13 x 100 mm tube. We vigorously mixed the gills and buffer solution for seconds. We transferred 20 μL buffer diluent from each set of gills to the surface of R2A agar plates. The agar plates were incubated at 20ºC for 48 to72 h. The resulting individual bacterial colonies were enumerated and the data were presented as the number of colony forming units per gram of gill (CFU/g). Bacterial colonies representing the predominant colony phenotypes were transferred to fresh R2A media and were characterized using standard methods for identification of bacteria. In statistical analyses of our data, we considered P<0.05 to be significant. We determined the LC50 for cisco fry following the three day treatment using the Trimmed Spearman Karber method. Because the Shapiro-Wilks test showed that data were not normally distributed, we used the non-parametric Kruskal-Wallis one-way analyses of variance test to examine differences in survival (response variable) among Chloramine T concentrations (independent variable) as well as the numbers of bacterial colonies present on the gills. When differences were significant, we used Tukey’s pairwise comparison test to determine which concentrations differed significantly in survival. RESULTS: Cisco fry ( mm) exposed to a one hour treatment of Chloramine T for three consecutive days had significantly lower survival (p=0.04, F Stat=2.94, DF=6) in concentrations greater than 80mg/L (Table 1). Using the observed survival rates, we calculated mean and the 95% confidence interval (CI) for LC50 to be 112 mg/L (95%CI= mg/L). In comparison of viable cell counts before and after the Chloramine T treatment at 20 mg/L for an hour for three consecutive days, the overall mean count for pretreated fry was 2.34 x 10 2 CFU/ml, whereas, the mean for treated fry was significantly higher at 1.19 x 10 4 (P=0.07, T Stat=1.91) (Figure 4). There were 115 bacterial isolates identified from untreated fry and 118 isolates identified from Chloramine T treated fry. We identified 47 species of bacteria on the outer surface of cisco gill filaments before and 46 species after treatment (Table 2). There were 13 strains that were eliminated and an additional 13 strains that appeared using the Chloramine T treatment. Bacteria recovered in greatest frequency (%) before (9.73, 8.85) and after treatment were Paracoccus sp. and Acinetobacter lwoffii (11.11, 10.26). Aeromonas sp. was identified after the Chloramine T treatment but comprised less than 1% of the bacterial community. RESULTS: Cisco fry ( mm) exposed to a one hour treatment of Chloramine T for three consecutive days had significantly lower survival (p=0.04, F Stat=2.94, DF=6) in concentrations greater than 80mg/L (Table 1). Using the observed survival rates, we calculated mean and the 95% confidence interval (CI) for LC50 to be 112 mg/L (95%CI= mg/L). In comparison of viable cell counts before and after the Chloramine T treatment at 20 mg/L for an hour for three consecutive days, the overall mean count for pretreated fry was 2.34 x 10 2 CFU/ml, whereas, the mean for treated fry was significantly higher at 1.19 x 10 4 (P=0.07, T Stat=1.91) (Figure 4). There were 115 bacterial isolates identified from untreated fry and 118 isolates identified from Chloramine T treated fry. We identified 47 species of bacteria on the outer surface of cisco gill filaments before and 46 species after treatment (Table 2). There were 13 strains that were eliminated and an additional 13 strains that appeared using the Chloramine T treatment. Bacteria recovered in greatest frequency (%) before (9.73, 8.85) and after treatment were Paracoccus sp. and Acinetobacter lwoffii (11.11, 10.26). Aeromonas sp. was identified after the Chloramine T treatment but comprised less than 1% of the bacterial community. CONCLUSION: The recommended dose for Chloramine T is 12-20mg/L administered for three treatments in the culture units. Our results suggest that is safe to treat cisco fry (15-20mm) at this level as it is well below the LC 50 value of 112mg/L. However, the bacterial presence at the recommended dose was observed to be confounding with an increase in the bacterial community. Additional bacterial species are able to colonize and proliferate on the gill surface of cisco fry following the recommended treatment. As a result managers must continue to take preventative measures for limiting the introduction of new bacterial species in addition to controlling bacterial gill disease. CONCLUSION: The recommended dose for Chloramine T is 12-20mg/L administered for three treatments in the culture units. Our results suggest that is safe to treat cisco fry (15-20mm) at this level as it is well below the LC 50 value of 112mg/L. However, the bacterial presence at the recommended dose was observed to be confounding with an increase in the bacterial community. Additional bacterial species are able to colonize and proliferate on the gill surface of cisco fry following the recommended treatment. As a result managers must continue to take preventative measures for limiting the introduction of new bacterial species in addition to controlling bacterial gill disease. INTRODUCTION:: Cisco population levels in Lake Ontario are currently low compared to historic abundance due to overharvesting and lack of adequate spawning habitat. Currently, there is an effort to re-establish cisco back into Lake Ontario by the U.S. Geological Survey (USGS) and the New York Department of Environmental Conservation (NYDEC).The Tunison Lab of Aquatic Science (TLAS) has developed culture techniques and protocols to rear cisco for re- introduction. Feeding larval cisco diets ( μm) increases water turbidity where fine particulates can irritate the gills and lead to bacterial infection. Chloramine T was approved by the US Food and Drug Administration to treat gill diseases associated with Flavobacterium spp. (Figure 1) but little was known about its effects on cisco larvae. The objectives of this study were to determine the LC 50 of Chloramine-T, and determine how the bacterial flora on the gills of larval cisco is impacted following a one hour treatment for three consecutive days. INTRODUCTION:: Cisco population levels in Lake Ontario are currently low compared to historic abundance due to overharvesting and lack of adequate spawning habitat. Currently, there is an effort to re-establish cisco back into Lake Ontario by the U.S. Geological Survey (USGS) and the New York Department of Environmental Conservation (NYDEC).The Tunison Lab of Aquatic Science (TLAS) has developed culture techniques and protocols to rear cisco for re- introduction. Feeding larval cisco diets ( μm) increases water turbidity where fine particulates can irritate the gills and lead to bacterial infection. Chloramine T was approved by the US Food and Drug Administration to treat gill diseases associated with Flavobacterium spp. (Figure 1) but little was known about its effects on cisco larvae. The objectives of this study were to determine the LC 50 of Chloramine-T, and determine how the bacterial flora on the gills of larval cisco is impacted following a one hour treatment for three consecutive days. Toxicity Evaluation In order to determine the correct dosage of Chloramine T to control bacterial growth we first evaluated the toxicity of the chemical to cisco. Three replicate groups of 25 fish (Figure 2) each (n = 75 fish per treatment) were exposed to one of seven treatments (0, 5, 10, 20, 40, 80, 160 mg/L) of Chloramine T. Concentrations were dispensed using a chicken waterer with the total volume (1.9 L) siphoned into the tank over an hour period for three consecutive days. Custom-made cylindrical containers (8 cm diameter, 4 cm high) with 1 mm nylon mesh on the base were suspended at the water surface on a metal screen to promote up-welling fresh water at 12 L/min. (Figure 3). The mean temperature, pH and total hardness of this water was 8.1ºC, 6.82, 230mg/L as calcium carbonate, respectively, and remained relatively constant. Survival of fish was recorded daily for four days following the completion of the third exposure treatment. Toxicity Evaluation In order to determine the correct dosage of Chloramine T to control bacterial growth we first evaluated the toxicity of the chemical to cisco. Three replicate groups of 25 fish (Figure 2) each (n = 75 fish per treatment) were exposed to one of seven treatments (0, 5, 10, 20, 40, 80, 160 mg/L) of Chloramine T. Concentrations were dispensed using a chicken waterer with the total volume (1.9 L) siphoned into the tank over an hour period for three consecutive days. Custom-made cylindrical containers (8 cm diameter, 4 cm high) with 1 mm nylon mesh on the base were suspended at the water surface on a metal screen to promote up-welling fresh water at 12 L/min. (Figure 3). The mean temperature, pH and total hardness of this water was 8.1ºC, 6.82, 230mg/L as calcium carbonate, respectively, and remained relatively constant. Survival of fish was recorded daily for four days following the completion of the third exposure treatment. METHOD: We captured adult cisco from Chaumont Bay, Lake Ontario in December and transported them to outside raceways at TLAS. Adult fish were spawned and the eggs were moved into our hatchery where they incubated under normal hatchery conditions. Once cisco hatched, larvae were transferred to circular tanks and allowed to grow for 50 days before experiments began. METHOD: We captured adult cisco from Chaumont Bay, Lake Ontario in December and transported them to outside raceways at TLAS. Adult fish were spawned and the eggs were moved into our hatchery where they incubated under normal hatchery conditions. Once cisco hatched, larvae were transferred to circular tanks and allowed to grow for 50 days before experiments began. Figure 4 : Bacterial cell counts (CFU/ml) on then outer gill surface before and after a one hour Chloramine T treatment for three consecutive days. Table 1 : Percent survival of cisco exposed to various concentrations of Chloramine T for one hour for three consecutive days. Table 2 : Bacterial species presence on the outer gill surface before and after a one hour Chloramine T treatment for three consecutive days. Figure 3 : Chloramine T toxicity evaluation jar set up. Figure 2 : Cisco fry ACKNOWLEDGEMENTS: A special thanks to Mike Connerton of the NYSDEC Cape Vincent, NY office for help collecting adult cisco to spawn. Thanks also to the Tunison Lab fish culture crew for their efforts raising cisco fry through the duration of the experiment. ACKNOWLEDGEMENTS: A special thanks to Mike Connerton of the NYSDEC Cape Vincent, NY office for help collecting adult cisco to spawn. Thanks also to the Tunison Lab fish culture crew for their efforts raising cisco fry through the duration of the experiment. Figure 1 : Flavobacterium spp.