Date of Approval: _______________ FREEDOM OF INFORMATION SUMMARY PMF 005-639 Hydrogen peroxide (35% w/w) “…for the control of mortality associated with bacterial gill disease on freshwater-reared salmonid finfish, and the control of egg mortality associated with saprolegniasis on all cold- and cool- freshwater-reared finfish eggs.” PUBLIC MASTER FILE Sponsored by: Upper Midwest Environmental Sciences Center United States Geological Survey I. GENERAL INFORMATION 1 II. EFFECTIVENESS 3 A. Dose Characterization 3 B. Substantial Evidence B-1. Freshwater-reared Finfish Effectiveness Clinical Field Trial, PMF 005-639 3 B-2. Freshwater-reared Finfish Egg Effectiveness Clinical Field Trial, PMF 005-639 4 B-3. Freshwater-reared Finfish Egg Effectiveness Clinical Field Trial, INAD 010-023 6 III. TARGET ANIMAL SAFETY 8 A. Freshwater-reared Finfish 8 A-1. Target Animal Safety Study, INAD 010-023 8 B. Freshwater-reared Finfish Eggs 13 B-1. Target Animal Safety Study, PMF 005-639 13 B-2. Target Animal Safety Study, PMF 005-639 14 B-3. Target Animal Safety Study, PMF 005-639 15 B-4. Target Animal Safety Study, INAD 010-023 16 IV. HUMAN SAFETY 19 A. Toxicity 19 B. Safe Concentration of Total Residues – Determination of No Observed Effect Level (NOEL) 19 C. Safe Concentration of Total Residues – Calculation of the Acceptable Daily Intake (ADI) and the Safe Concentration (SC) 19 D. Total Residue Depletion and Metabolism Studies 19 E. Comparative Metabolism Studies 19 F. Residue Depletion Studies 19 G. Tolerance and Withdrawal Time 19 H. Regulatory Method for Residues 19 I. User Safety Concerns 19 V. AGENCY CONCLUSIONS 20 VI. APPROVED LABELING 21 I. GENERAL INFORMATION File Number: PMF 005-639 Sponsor: Upper Midwest Environmental Sciences Center United States Geological Survey 2630 Fanta Reed Road La Crosse, Wisconsin 54603 Established Name: Hydrogen peroxide Proprietary Name: To be determined Dosage Form: Liquid solution How Supplied: 5-gallon and 55-gallon containers How Dispensed: Over-the-counter (OTC) Amount of Active Ingredients: 35% w/w (weight in water) Route of Administration: Immersion Species/Class: Freshwater-reared finfish, and freshwater-reared finfish eggs Recommended Dosage: Freshwater-reared finfish: Species Dose Duration Freshwater-reared salmonids, largemouth bass, and muskellunge 50-100 mg/L 60 minutes 100 mg/L 30 minutes Pallid sturgeon, walleye, white sucker, bluegill, channel catfish, fathead minnow, yellow perch and all other freshwater-reared finfish (except northern pike & paddlefish)* 50-75 mg/L 60 minutes Walleye, white sucker, bluegill, channel catfish, fathead minnow, yellow perch and all other freshwater-reared finfish fry (except northern pike, pallid sturgeon & paddlefish)* 50 mg/L 60 minutes *Initial bioassay recommended Freshwater-reared finfish eggs: 500 to 1000 mg/L for 15 minutes in a continuous flow system every other day or on every day until hatch for all cold- or cool- freshwater- reared finfish eggs or 750 to 1000 mg/L for 15 minutes in a continuous flow system every other day or every day until hatch for all warm- freshwater-reared finfish eggs. Pharmacologic Category: Topical antiseptic and cleansing agent Indications: Hydrogen peroxide (35% w/w) is indicated for the control of mortality associated with bacterial gill disease on freshwater-reared salmonid finfish, the control of mortality associated with external columnaris on all cool- freshwater- reared finfish and freshwater-reared channel catfish, and the control of egg mortality associated with saprolegniasis on all freshwater-reared finfish eggs. II. EFFECTIVENESS A. Dose Characterization: The primary effect of hydrogen peroxide results from localized action at the topical site of administration. The concentration of the active drug at the topical site is a function of the administered concentration, exposure period, and water conditions. These three conditions and the sensitivity of the pathogen to the drug are considered the primary determinants of effectiveness. B. Substantial Evidence: Data contained in the sponsor’s public master file (PMF 005-639) and investigational new animal drug file (INAD 010-023) demonstrate the effectiveness of hydrogen peroxide at specific concentrations and exposure times for the control of mortality associated with bacterial gill disease on freshwater-reared salmonid finfish, and for the control of egg mortality associated with saprolegniasis on all cold- and cool- freshwater-reared finfish eggs. 1. Freshwater-reared Finfish Effectiveness, Clinical Field Trial, PMF 005-639: a. Title: “Pivotal Studies to Evaluate the Efficacy of Hydrogen Peroxide to Control Mortalities Associated with External Flavobacter Infections on Cultured Fish at Selected Fish Hatcheries” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the effectiveness of hydrogen peroxide as a static bath at concentrations of 0, 57, 113, and 226 mg/L for 30 minutes (rainbow trout) and 60 minutes (brown trout and chinook salmon) every other day for three treatments to control mortality in fish naturally infected with filamentous bacteria as diagnosed microscopically at necropsy. ii. Test Animals: Young brown trout (Salmo trutta) (9 g), Chinook salmon (Oncorhynchus tschawytscha) (3.5 g), and rainbow trout (Oncorhynchus mykiss) (10 g) were used. All fish were cultured in outdoor raceways and were naturally infected with bacterial gill disease. After disease diagnosis, fish were removed from the source raceway and hand counted according to random assignment into each test tank (brown trout, n=20; chinook salmon, n=44; rainbow trout, n=28). Four treatment groups were used for each species: 0, 57, 113, and 226 mg/L. iii. Controls: Infected, untreated finfish. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion, static bath. vi. Study Duration: 5 day treatment period followed by a 12 to 14 day post treatment observation period. vii. Parameters Studied: Percent mortality, gill condition, hydrogen peroxide concentration, dissolved oxygen, temperature and pH. viii. Results: Cumulative mortality at the end of 12 or 14 days is represented in Table II.B.1: TABLE II.B.1 Cumulative Mortality (%) at 12 (a) or 14 (b) Days H2O2 Concentration (mg/L) Brown Trout (a) (60-minute bath) Chinook Salmon (b) (60-minute bath) Rainbow Trout (b) (30-minute bath) 0 11.7 21.2 31.0 57 0.0 7.6 25.0 113 6.7 22.0 14.3 226 36.7 72.0 7.1 ix. Conclusions: The data in this study support a minimum efficacious dose of 57 mg/L administered as a 60 minute static bath, or 113 mg/L administered as a 30 minute static bath, every other day for three treatments for the control of mortality associated with bacterial gill disease in freshwater-reared salmonids. Mortality among brown trout and Chinook salmon in the 226 mg/L group is consistent with dose-related toxicity (see Target Animal Safety). 2. Freshwater-reared Finfish Effectiveness, Clinical Field Trial, INAD 10- 023: a. Title: “Efficacy of hydrogen peroxide to control mortality associated with external columnaris on channel catfish fingerings (Ictalurus punctatus).” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the effectiveness of hydrogen peroxide as a static bath at concentrations of 0, 50, 75 or 100 mg/L for 60 min every other day for three treatments to control mortality associated with external columnaris on channel catfish fingerlings. ii. Test Animals: Young channel catfish (Ictalurus punctatus; 2.28 g) were used. Hatchery personnel observed channel catfish fingerlings exhibiting behavior characteristic of a columnaris infection and mortality began to increase. Fish were diagnosed with external columnaris by UMESC and hatchery personnel. iii. Controls: Infected, untreated channel catfish. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion, static bath. vi. Study Duration: 5 day treatment period followed by a 10 day post treatment observation period. vii. Parameters Studied: Percent mortality, hydrogen peroxide concentration, dissolved oxygen, temperature, and pH. viii. Results: Cumulative percent mortality at the end of 15 days is represented in Table II.B.2: TABLE II.B.2 Cumulative mean percent mortality at 15 Days H2O2 Concentration (mg/L) Channel Catfish 0 80.8 50 47.7 75 28.2 100 41.0 x. Conclusions: The data in this trial support an efficacious treatment range of 50 to 75 mg/L administered for 60 min on three alternate days to control mortality associated with external columnaris on channel catfish. Results of this study support the use of hydrogen peroxide treatment to control mortalities associated with external columnaris on channel catfish. 3. Freshwater-reared Finfish Effectiveness, Non-clinical Trial, INAD 10- 023: a. Title: “Efficacy of Hydrogen Peroxide to Control Mortality Associated with Saprolegniasis Infections on Channel Catfish” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the effectiveness of hydrogen peroxide as a static bath at concentrations of 0, 75 or 100 mg/L for 60 min every other day for three treatments to control mortality associated with saprolegniasis on channel catfish fingerlings. ii. Test Animals: Young channel catfish (Ictalurus punctatus; 4.2 – 14.7 g) were used. Fish were experimentally infected by first abrading a ~1-cm2 area on the left side of the fish followed by immersion challenge in a static tank containing Saprolegnia parasitica zoospores. Pure S. parasitica cultures were prepared on sterile hemp seeds; hemp seeds with visible fungal growth were then suspended in the challenge bath for 24-h prior to fish immersion challenge. After abrasion, the fish were immersed in the challenge tank for 6 to 24 h at 13 + 2 °C. Fish were hand counted from the immersion challenge bath to aquaria according to a random number table until each aquarium had 10 fish. iii. Controls: Infected, untreated channel catfish. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion, static bath. vi. Study Duration: Four separate trials were conducted. Each trial consisted of an infection period, a 5 day treatment period, and a 10 day post treatment observation period for a total duration of 16 days. The 24-h immersion challenge used in Trials 1 and 2 resulted in nearly 100% mortality that resulted in the early termination of these trials. Results from Trials 1 and 2 are not included in this FOI. vii. Parameters Studied: Percent mortality, hydrogen peroxide concentration, dissolved oxygen, temperature, and pH. viii. Results: Cumulative percent mortality in Trials 3 and 4 at the end of 15 days is represented in Table II.B.3: TABLE II.B.3 Cumulative mean percent mortality at 15 Days H2O2 Concentration (mg/L) Trial 3 (12 h infection) Trial 4 (6 h infection) 0 100 87 75 87 37 100 90 NA xi. Conclusions: The results of this study indicate that hydrogen peroxide applied at 75 mg/L for 60 min once every-other-day for three treatments reduce mortality and increase survival of channel catfish fingerlings infected with Saprolegnia parasitica. Results of this study support the use of hydrogen peroxide to control mortality associated with fungal infections on channel catfish fish. 4. Freshwater-reared Finfish Effectiveness, Clinical Field Trial, INAD 10-023: a. Title: “Efficacy of hydrogen peroxide to control mortality associated with external columnaris on walleye fingerings (Stizostedion vitreum).” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the effectiveness of hydrogen peroxide as a static bath at concentrations of 0, 50, 75 or 100 mg/L for 60 min every other day for three treatments to control mortality associated with external columnaris on walleye fingerlings. ii. Test Animals: Young walleye (Sander vitreum; 12 g) were used. Fish were diagnosed with external columnaris by UMESC and hatchery personnel. iii. Controls: Infected, untreated finfish. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion, static bath. vi. Study Duration: 5 day treatment period followed by a 10 day post treatment observation period. vii. Parameters Studied: Percent mortality, hydrogen peroxide concentration, dissolved oxygen, temperature, and pH. viii. Results: Cumulative mortality (%) at the end of 15 days is represented in Table II.B.4: TABLE II.B.4 Cumulative mortality (%) at day 15 H2O2 Concentration (mg/L) Walleye 0 80.8 50 47.4 75 28.2 100 41.0 xii. Conclusions: The data from this study demonstrate that treatment regimens of 50 – 75 mg/L administered for 60 min on three alternate days are efficacious in reducing mortality of walleye fingerlings infected with external columnaris. Results of this study and other published studies support the use of hydrogen peroxide treatment for the control of external columnaris on all cool- freshwater-reared finfish. 5. Freshwater-reared Finfish Effectiveness, Supplemental Efficacy Data: a. Title: “Efficacy of Hydrogen Peroxide to Control Mortalities Associated with Columnaris Infections on Yellow Perch.” The data were submitted to CVM (PMF 5639, February 2000). b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: vii. The primary purpose of the study was to evaluate the effectiveness of hydrogen peroxide (35% w/w) as a static bath at concentrations of 0, 50 or 100 mg/L for 60 min every other day for three treatments to control mortality associated with external flavobacterial on yellow perch fingerlings. Yellow perch (Perca flavescens; 1 g) with a natural flavobacterial infection were used. Fish were evenly distributed among the test tanks until each tank had 174 fish with three replicates per treatment group. An infected, untreated yellow perch control group was used. The trial consisted of a 5 day treatment period and a 9 day post treatment observation period for a total duration of 14 days. Cumulative percent mortality is represented in Table II.B.5. Hydrogen peroxide controlled mortalities associated with flavobacterial infections on yellow perch. The optimum treatment regimen was 50 mg/L for 60 minutes. TABLE II.B.5 Cumulative mean percent mortality H2O2 Concentration (mg/L) Cumulative percent mortality 0 7.5 50 1.1 100 5.2 6. Freshwater-reared Finfish Effectiveness, Supplemental Efficacy Data: a. Title: “Use of Hydrogen Peroxide to Treat Eperimentally Induced Bacterial Gill Disease in Rainbow Trout”. The manuscript from which the data are drawn was submitted to CVM (PMF 5639, February 2000) b. Investigator: J.S. Lumsden Fish Pathology Laboratory University of Guelph Guelph, Ontario N1G 2W1, Canada c. Study design and results: The primary study objective was to determine the efficacy of hydrogen peroxide to control mortality associated with experimentally induced bacterial gill disease (BGD) caused by Flavobacterium branchiophilium on rainbow trout Oncorhynchus mykiss. Rainbow trout 12 – 20 g) were experimentally infected by immersion in a static tank containing Flavobacterium branchiophilium at a concentration of 1 x 105 colony-forming units (CFU)/mL for 1 h. Flavobacterium branchiophilium challenge concentrations were verified by plate count. Experimental treatments were administered in the same tanks where experimental infections were conducted. Fish were initially stocked at 100 g/L into experimental tanks, resulting in between 50 to 150 fish per tank. Hydrogen peroxide treatments were administered as a static bath at concentrations of 0, 25, 75 100, 125, 175, or 250 mg/L for 60 min every other day for two or three treatments to control mortality associated with experimentally induced bacterial gill disease caused by Flavobacterium branchiophilium on rainbow trout fingerlings. Three separate experiments were conducted. Each trial consisted of an infection period, a 48 h pre-treatment period followed by a 3 to 5 day treatment period, followed by a post-treatment observation period. The post-treatment observation period lasted at least two days after the last mortality. Cumulative percent mortality at the end of the observation period is represented in Table II.B.6. The results of this study indicate that hydrogen peroxide applied at 25 to 250 mg/L for 60 min once every-other-day for two to three treatments reduce mortality and increase survival of rainbow trout fingerlings infected with Flavobacterium branchiophilium. Results of this study support the use of hydrogen peroxide to control mortality associated with bacterial gill disease on trout. The data are summarized in Lumsden, J.S., V.E. Ostland, and H.W. Ferguson. 1998. Use of Hydrogen Peroxide to Treat Experimentally Induced Bacterial Gill Disease in Rainbow Trout. Journal of Aquatic Animal Health 10:230240. TABLE II.B.6 Cumulative mean percent mortality of rainbow trout experimentally infected with bacterial gill disease. H2O2 Concentration (mg/L) Experiment 1 Experiment 2 Experiment 3 0 44.4 61.1 60.0 25 34.1 ND 4.2 75 ND ND 7.5 100 25.9 21.1 ND 125 ND ND 13.3 175 ND ND 9.2 250 2.2 15.5 24.2 7. Freshwater-reared Finfish Egg Effectiveness, Clinical Field Trial, PMF 005-639: a. Title: “Efficacy of Hydrogen Peroxide Treatments to Control Saprolegniasis Infections on Salmonid Eggs.” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the effectiveness of hydrogen peroxide administered at concentrations of 113, 283, 566, and 1,132 mg/L for 15 minutes every other day until hatch to control and prevent fungal infections caused by Saprolegniaceae on the eggs of rainbow trout. ii. Test Animals: Non-eyed fertilized eggs, 36-hours old, of rainbow trout (Oncorhynchus mykiss). Eggs were assigned to three test groups consisting of 500 eggs for each treatment regimen in two trials. Trial 1 administered 0, 113, 283 or 566 mg/L; and Trial 2 administered 0 or 1,132 mg/L hydrogen peroxide in 15 minute exposures every other day until eggs hatched. Studies in both trials were conducted on groups with induced (at 10% of eggs visibly infected) and non-induced (0% of eggs visibly infected) S. parasitica infection rates at the start of treatment. iii. Controls: Uninfected, untreated eggs and infected, untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a constant-flow system. vi. Study Duration: 53 days. vii. Parameters Studied: Percent of eggs that hatched, percent fungal infection of eggs, hydrogen peroxide concentration, pH, dissolved oxygen, and temperature. viii. Results: Table II.B.5 describes the mean infection rate and the mean percent hatch for 0, 113, 283, and 566 mg/L (Trial 1) and 0 and 1,132 mg/L (Trial 2). TABLE II.B.5: Percent Hatch and Percent Infection H2O2 Concentration (mg/L) Trial Number Mean Infection (%) Mean Hatch (%) Initial Final Uninfected Eggs 0 1 0.0 1.2 95.6 113 1 0.0 0.0 95.1 283 1 0.0 1.2 95.5 566 1 0.0 0.7 95.3 0 2 0.0 3.0 90.6 1,132 2 0.0 0.0 90.6 Infected Eggs 0 1 10.0 54.1 50.0 113 1 10.0 49.3 56.1 283 1 10.0 38.5 64.6 566 1 10.0 25.7 80.0 0 2 10.0 67.9 27.3 1,132 2 10.0 29.0 68.3 ix. Conclusions: The data show a significant increase in the mean percent hatch of infected, treated eggs compared with infected, untreated eggs. These data support the effectiveness of hydrogen peroxide for the control of egg mortality associated with saprolegniasis on salmonid eggs when applied at doses of 566 to 1,132 mg/L for 15 minutes every other day until hatch. 6. Freshwater-reared Finfish Egg Effectiveness, Clinical Field Trial, INAD 010-023: a. Title: “Efficacy of Hydrogen Peroxide to Control Mortality Associated with Saprolegniasis on Paddlefish Polyodon spathula, Walleye Stizostedion vitreum, and White Sucker Catostomus commersoni Eggs.” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To determine the effectiveness of hydrogen peroxide to control egg mortality (as measured by the probability of egg hatch) associated with fungal infections on cool- and warmwater fish eggs. ii. Test Animals: Non-eyed eggs of walleye, white sucker, and paddlefish. Eggs were assigned to four test groups consisting of a 30 mL volume of eggs for each treatment regimen. Hydrogen peroxide was administered every other day for 15 minute exposures of 0, 283, 565, or 1,130 mg/L until eggs hatched. Eggs developed a natural infection of saprolegniasis as determined by the fish culturist. iii. Controls: Infected, untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a constant-flow system. vi. Study Duration: 60 days. vii. Parameters Studied: Mean percent hatch, hydrogen peroxide concentration, pH, dissolved oxygen, and temperature. viii. Results: Table II.B.6 describes the mean percent hatch for 0, 283, 565, and 1,130 mg/L treatments. ix. TABLE II.B.6: Mean Percent Hatch Species Mean Percent Hatch (p value vs. control) 0 283 mg/L 565 mg/L 1,130 mg/L Walleye 11% 55% (p<0.01) 49% (p=0.01) 65% (p<0.01) White Sucker 9% 23% (p<0.01) 47% (p<0.01) 69% (p<0.01) Paddlefish 1% 6% (p=0.06) 5% (p=0.08) 24% (p<0.01) x. Conclusions: The data show a significant increase in the mean percent hatch of infected, treated eggs compared with infected, untreated eggs. These data support the effectiveness of hydrogen peroxide for the control of egg mortality associated with saprolegniasis on cool- and warmwater finfish eggs at doses of 283 to 1,130 mg/L for 15 minutes every other day until hatch. 7. Freshwater-reared Finfish Egg Effectiveness, Clinical Field Trial, INAD 010-023: a. Title: “Efficacy of hydrogen peroxide to control mortality associated with saprolegniasis in channel catfish eggs (Ictalurus punctatus).” b. Investigator: Jeffrey J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To determine the effectiveness of hydrogen peroxide to control egg mortality (as measured by the probability of egg hatch) associated with fungal infections on channel catfish eggs. ii. Test Animals: Non-eyed eggs of channel catfish. Eggs were assigned to three test groups consisting of a 0.79 kg mass of eggs per test chamber for each treatment regimen. Approximately 0.15 kg masses of eggs were weighed then distributed to each test chamber until each chamber had received a total mass of 0.79 kg (equivalent to 17,500 eggs). Hydrogen peroxide was administered every day for 15 minute exposures of 0, 500, or 1,000 mg/L until eggs hatched. Eggs developed a natural infection of saprolegniasis as confirmed by laboratory culture and analysis of cultured zoospore reproductive morphology. iii. Controls: Infected, untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a constant-flow system. vi. Study Duration: 6 days. vii. Parameters Studied: Mean percent hatch, hydrogen peroxide concentration, pH, dissolved oxygen, temperature, and total hardness and alkalinity. x. Results: Table II.B.6 describes the mean percent hatch for 0, 500, and 1,000 mg/L treatments. xi. TABLE II.B.7: Mean Percent Hatch Concentration (mg/L) Mean Percent Hatch 0 44 500 54 750 69 x. Conclusions: The data show a significant increase in the mean percent hatch of infected, treated eggs compared with infected, untreated eggs. Results of this study and other published studies support the effectiveness of hydrogen peroxide for the control of egg mortality associated with saprolegniasis on warm freshwater-reared finfish eggs at doses of 750 to 1,000 mg/L for 15 minutes every day until hatch. III. TARGET ANIMAL SAFETY Data in contained in the sponsor’s public master file (PMF 005-639) and investigational new animal drug file (INAD 010-023) demonstrate the safety of hydrogen peroxide at specific concentrations and exposure times for the treatment of finfish and finfish eggs in a wide range of cold-, cool-, and warmwater fishes. The requirements for target animal safety for all freshwater-reared finfish at the specified doses have been satisfied. An initial bioassay is recommended for species not specifically tested and for northern pike and paddlefish adults, and for northern pike, pallid sturgeon, and paddlefish fry. The requirements for target animal safety for cold- and cool- freshwater-reared finfish eggs at the specified doses have been satisfied. Additionally, INAD 010- 023 contains data that address the safety of hydrogen peroxide in the treatment of non-eyed and eyed salmonid eggs. The data in these studies indicate there is a period of time during development, Day 6 through Day 10 (78 to 135 DTU?C), in which rainbow trout embryos may be sensitive to treatments with hydrogen peroxide within the recommended dose range. Treatment for this species, at this developmental time point, should be avoided. A. Freshwater-reared Finfish: 1. Target Animal Safety Study, INAD 010-023: a. Title: “Toxicity Assessment of Hydrogen Peroxide to Cold-, Cool-, and Warmwater Fish.” b. Investigator: Mark P. GaikowskiU.S. Geological Survey Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To determine the species most sensitive to 60 or 180 minute hydrogen peroxide bath treatments by exposing the fry and/or fingerling life stage of one or more fish species from each of the major families cultured by public aquaculture (Salmonidae, Esocidae, Percidae, Ictaluridae, Catostomidae, Centrarchidae, Cyprinidae, Percichthyidae, Acipenseridae, and Polyodontidae). ii. Test Animals: Fingerling and fry rainbow trout (Oncorhynchus mykiss), lake trout (Salvelinus namaycush), Atlantic salmon (Salmo salar), northern pike (Esox lucius), muskellunge (Esox masquinongy), walleye (Stizostedion vitreum), yellow perch (Perca flavescens), channel catfish (Ictalurus punctatus), largemouth bass (Micropterus salmoides), bluegill (Lepomis macrochirus), white sucker (Catostomus commersoni), fathead minnow (Pimephales promelas), pallid sturgeon (Scaphirhynchus albus), paddlefish (Polyodon spathula) were obtained from various state and federal hatcheries. Test animals were fed a diet appropriate for their species and life stage consisting of either: salmon feed, forage, brine shrimp, or trout feed. Test animals were maintained at appropriate temperatures and culture conditions for their species and life stage. Five treated and one non-treated group(s) were used for each species and life stage. The concentrations varied depending on an initial range finding study with differing amounts for the 60 and 180 minute exposure times. The amounts were selected such that therapeutic and lethal concentrations would be contained within the range. Survival curves were generated for each exposure time. Fish were exposed to a total of three static baths of hydrogen peroxide administered once every other day. Exposures were administered for 60 and 180 minutes. Each treatment group was replicated 3 times. Refer to Table III.A.1.1: Table III.A.1.1: Experimental Design and Treatment Animals Species Hydrogen Peroxide Concentration (mg/L) Exposure time Atlantic salmon fingerling 0, 150, 250, 417, 695, 1158 60 minutes 0, 49, 81, 136, 226, 376 180 minutes Rainbow trout fry 0, 212, 354, 706, 1413, 2825 60 minutes 0, 88, 147, 244, 407, 678 180 minutes Rainbow trout fingerling 0, 183, 305, 509, 848, 1413 60 minutes 0, 92, 153, 254, 424, 706 180 minutes Lake trout fingerling 0, 337, 562, 936, 1559, 2599 60 minutes 0, 46, 77, 128, 212, 354 180 minutes Northern pike fry 0, 111, 185, 310, 515, 859 60 minutes 0, 36, 61, 102, 170, 283 180 minutes Northern pike fingerling 0, 86, 144, 238, 398, 663 60 minutes 0, 36, 61, 102, 170, 283 180 minutes Muskellunge fry 0, 118, 195, 325, 542, 904 60 minutes 0, 36, 61, 102, 170, 283 180 minutes Muskellunge fingerling 0, 118, 195, 325, 542, 904 60 minutes 0, 53, 88, 147, 244, 407 180 minutes Walleye fry 0, 29, 49, 81, 136, 226 60 minutes 0, 18, 29, 49, 81, 136 180 minutes Walleye fingerling 0, 24, 40, 66, 108, 181 60 minutes 0, 11, 19, 32, 53, 88 180 minutes Yellow perch fry 0, 32, 53, 88, 147, 244 60 minutes 0, 10, 17, 28, 47, 79 180 minutes Yellow perch fingerling 0, 88, 147, 244, 407, 678 60 minutes 0, 53, 88, 147, 244, 407 180 minutes Channel catfish fry 0, 53, 88, 147, 244, 407 60 minutes 0, 19, 32, 53, 88, 147 180 minutes Channel catfish fingerling 0, 53, 88, 147, 244, 407 60 minutes 0, 19, 32, 53, 88, 147 180 minutes Largemouth bass fry 0, 121, 202, 337, 562, 936 60 minutes 0, 103, 171, 285, 475, 791 180 minutes Largemouth bass fingerling 0, 53, 88, 147, 244, 407 60 minutes 0, 53, 88, 147, 244, 407 180 minutes Bluegill fry 0, 53, 88, 147, 244, 407 60 minutes 0, 32, 53, 88, 147, 244 180 minutes Bluegill fingerling 0, 32, 53, 88, 147, 244 60 minutes 0, 32, 53, 88, 147, 244 180 minutes White sucker fry 0, 53, 88, 147, 244, 407 60 minutes 0, 19, 32, 53, 88, 147 180 minutes White sucker fingerling 0, 88, 147, 244, 407, 678 60 minutes 0, 53, 88, 147, 244, 407 180 minutes Fathead minnow fry 0, 19, 32, 53, 88, 147 60 minutes 0, 11, 19, 32, 53, 88 180 minutes Fathead minnow fingerling 0, 53, 88, 147, 244, 407 60 minutes 0, 32, 53, 88, 147, 244 180 minutes Pallid sturgeon fry 0, 129, 215, 366, 610, 1017 60 minutes 0, 32, 53, 88, 147, 244 180 minutes Pallid sturgeon fingerling 0, 105, 175, 293, 488, 814 60 minutes 0, 32, 53, 88, 147, 244 180 minutes Paddlefish fingerling 0, 73, 122, 203, 339, 565 60 minutes Not tested 180 minutes iii. Controls: Untreated finfish of each species. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion, static bath. vi. Study Duration: 8 days. vii. Parameters Studied: Mortality was recorded at 0, 24, 48, 72, 96, 120, 144, 168, and 192 hours after the first exposure. At 96 and 192 hours, three fish were removed from the control and each of the treatment aquaria in the three lowest concentrations, euthanized, and the second gill arch collected and fixed in 2.5% glutaraldehyde/ 2% formaldehyde solution. Samples from the 60 minute time point were examined histologically. Dissolved oxygen, temperature, and pH were recorded daily in each test tank. viii. Results: Mean percent mortality by species, life stage, hydrogen peroxide concentration and exposure time are shown in Table III.A.1.2: Table III.A.1.2: Mean Mortality (%) at H2O2 Concentrations (mg/L) Species Mean Mortality (%) / H2O2 Conc. (mg/L) Exposure time Atlantic salmon fingerling 7/0, 7/150, 0/250, 53/417, 100/695, 100/1158 60 minutes 0/0, 0/49, 0/81, 0/136, 67/226, 100/376 180 minutes Rainbow trout fry 0/0, 7/212, 67/354, 100/706, 100/1413, 100/2825 60 minutes 0/0, 20/88, 87/147, 100/244, 100/407, 100/678 180 minutes Rainbow trout fingerling 0/0, 4/183, 12/305, 96/509, 100/848, 100/1413 60 minutes 0/0, 0/92, 58/153, 100/254, 100/424, 100/706 180 minutes Lake trout fingerling 0/0, 13/337, 100/562, 100/936, 100/1559, 100/2599 60 minutes 0/0, 0/46, 0/77, 13/128, 67/212, 100/354 180 minutes Northern pike fry 30/0, 27/111, 50/185, 93/310, 100/515, 100/859 60 minutes 17/0, 33/36, 30/61, 62/102, 100/170, 100/283 180 minutes Northern pike fingerling 4/0, 37/86, 100/144, 100/238, 100/398, 100/663 60 minutes 0/0, 12/36, 75/61, 100/102, 100/170, 100/283 180 minutes Muskellunge fry 4/0, 0/118, 62/195, 71/325, 100/542, 100/904 60 minutes 0/0, 0/36, 8/61, 88/102, 100/170, 100/283 180 minutes Muskellunge fingerling 0/0, 0/118, 25/195, 75/325, 100/542, 100/904 60 minutes 0/0, 0/53, 0/88, 29/147, 100/244, 100/407 180 minutes Walleye fry 0/0, 0/29, 0/49, >20/81, 67/136, 100/226 60 minutes 0/0, 0/18, 0/29, 0/49, 7/81, 100/136 180 minutes Walleye fingerling 4/0, 0/24, 0/40, 0/66, >20/108, 93/181 60 minutes 8/0, 0/11, 4/19, 0/32, 13/53, 80/88 180 minutes Yellow perch fry 0/0, 13/32, 13/53, 40/88, 100/147, 100/244 60 minutes 0/0, 7/10, 0/17, 20/28, 7/47, 60/79 180 minutes Yellow perch fingerling 0/0, 0/88, 12/147, 71/244, 93/407, 100/678 60 minutes 0/0, 0/53, 0/88, 50/147, 93/244, 100/407 180 minutes Channel catfish fry 0/0, 0/53, 7/88, 87/147, 100/244, 100/407 60 minutes 0/0, 0/19, 0/32, 31/53, 100/88, 100/147 180 minutes Channel catfish fingerling 0/0, 0/53, 17/88, 87/147, 100/244, 100/407 60 minutes 0/0, 0/19, 0/32, 12/53, 100/88, 100/147 180 minutes Largemouth bass fry 0/0, 0/121, 0/202, 67/337, 100/562, 100/936 60 minutes 0/0, 0/103, 93/171, 100/285, 100/475, 100/791 180 minutes Largemouth bass fingerling 4/0, 0/53, 4/88, 8/147, 87/244, 67/407 60 minutes 0/0, 0/53, 42/88, 100/147, 100/244, 100/407 180 minutes Bluegill fry 0/0, 0/53, 0/88, 80/147, 100/244, 100/407 60 minutes 0/0, 0/32, 0/53, 40/88, 100/147, 100/244 180 minutes Bluegill fingerling 0/0, 0/32, 0/53, 12/88, 77/147, 67/244 60 minutes 0/0, 0/32, 0/53, 87/88, 100/147, 100/244 180 minutes White sucker fry 7/0, 0/53, 60/88, 100/147, 100/244, 100/407 60 minutes 7/0, 7/19, 7/32, 33/53, 93/88, 100/147 180 minutes White sucker fingerling 0/0, 4/88, 67/147, 100/244, 100/407, 100/678 60 minutes 0/0, 4/53, 73/88, 100/147, 100/244, 100/407 180 minutes Fathead minnow fry 0/0, 0/19, 0/32, 0/53, 60/88, 100/147 60 minutes 0/0, 0/11, 0/19, 0/32, 93/53, 100/88 180 minutes Fathead minnow fingerling 0/0, 0/53, 12/88, 96/147, 100/244, 100/407 60 minutes 0/0, 0/32, 0/53, 96/88, 100/147, 100/244 180 minutes Pallid sturgeon fry 4/0, 67/129, 67/215, 100/366, 100/610, 100/1017 60 minutes 0/0, 4/32, 50/53, 100/88, 100/147, 100/244 180 minutes Pallid sturgeon fingerling 0/0, 4/105, 42/175, 100/293, 100/488, 100/814 60 minutes 0/0, 0/32, 0/53, 96/88, 100/147, 100/244 180 minutes Paddlefish fingerling 0/0, 73/73, 100/122, 100/203, 100/339, 100/565 60 minutes Not tested 180 minutes ix. Conclusions: The data in this study demonstrate the target animal safety of hydrogen peroxide administered to finfish as a 60 minute static bath treatment at the following dosages: freshwater-reared salmonids, largemouth bass and muskellunge up to 100 mg/L; pallid sturgeon, walleye, white sucker, bluegill, channel catfish, fathead minnow, yellow perch and all other freshwater-reared finfish (except northern pike & paddlefish) up to 75 mg/L; walleye, white sucker, bluegill, channel catfish, fathead minnow, yellow perch and all other freshwater-reared finfish fry (except northern pike & paddlefish) up to 50 mg/L. The data in this study also demonstrate the target animal safety of hydrogen peroxide administered to finfish at the following point dose as a 30 minute continuous flow treatment: freshwater-reared salmonids, large mouth bass and muskellunge only may be treated at 100 mg/L. B. Freshwater-Reared Finfish Eggs 1. Target Animal Safety Study, PMF 005-639: a. Title: “Safety of Hydrogen Peroxide Treatments on Fish Eggs.” b. Investigator: Jeffrey J. RachUpper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the safety of hydrogen peroxide as a water treatment at concentrations of 566, 1,132, 3,396, or 6,792 mg/L daily (Monday through Friday) for 15 minutes until hatch to control and prevent fungal infections caused by saprolegniasis on the eggs of freshwater-reared finfish. ii. Test Animals: One to 3-day-old eggs of walleye (Stizostedion vitreum), yellow perch (Perca flavescens), white sucker (Catostomus commersoni), lake sturgeon (Acipenser fulvescens), paddlefish (Polyodon spathula), common carp (Cyprinus carpio), and channel catfish (Ictalurus punctatus) were allocated in 30 mL volumes to control groups and treatment groups receiving 1,132, 3,396, and 6,792 mg/L. Eggs of northern pike (Esox lucius) were allocated in the same manner, except a 566 mg/L treatment group was substituted for the 6,792 mg/L treatment group used in the other species. Each group was replicated 3 times. Each group was treated for 15 minutes once daily (Monday through Friday) until all viable eggs had hatched. iii. Controls: Untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a continuous-flow system. vi. Study Duration: 110 days, inclusive of all species. vii. Parameters Studied: Mean percent hatch, water flow, temperature, dissolved oxygen, pH, water hardness, hydrogen peroxide concentration. viii. Results: Refer to Table III.B.1: Table III.B.1: Mean Percent Hatch by Treatment Concentration of H2O2 (mg/L) Species 0 566 1,132 3,396 6,792 Northern pike 32 32 37 34 - a Walleye 0 b - a 77 61 5 Yellow perch 59 b - a 100 c 66 18 White sucker 15 b - a 61 42 0 Lake sturgeon 51 b - a 57 c 61 c 40 Paddlefish 72 b - a 82 c 53 42 d Common carp 6 b - a 59 53 48 Channel Catfish 19 b - a 78 68 0 a Not tested. b Fungus observed on eggs. c (n=2) One replicate was not used in statistical analysis due to aquarium overflow. d All fry died after post hatch treatments. ix. Conclusions: The data in this study support the target animal safety of hydrogen peroxide at dosages of 566 to 1,132 mg/L for 15 minutes daily for eggs of northern pike, lake sturgeon and common carp. 2. Target Animal Safety Study, PMF 005-639: a. Title: “Hydrogen Peroxide Treatment Toxicity to Rainbow Trout Eggs. Part I: Safety.” b. Investigator: Mark P. Gaikowski Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To evaluate the safety of hydrogen peroxide as a water treatment at concentrations of 0, 566, 1,132, and 3,396 mg/L for 15 minutes daily (Monday through Friday) from fertilization until 5 days after hatch on the eggs and fry of rainbow trout (Oncorhynchus mykiss) and steelhead trout (Oncorhynchus mykiss, Skamania and Ganaraska strains). ii. Test Animals: Non-eyed, fertilized eggs, approximately 36 hours old, from 3 lots of rainbow trout and 2 lots of steelhead trout. 30 mL of eggs were assigned to each control and treatment group. Three replicates at each concentration were performed. iii. Controls: Untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a continuous-flow system. vi. Study Duration: 50-60 days. vii. Parameters Studied: Percent hatch, temperature, dissolved oxygen, pH, hydrogen peroxide concentration. viii. Results: Hydrogen peroxide treatments reduced the probability of hatch in a dose-dependent manner. Refer to Table III.B.2: Table III.B.2: Mean Hatch (%) by Hydrogen Peroxide Concentration (mg/L) Species 0 566 1,132 3,396 Rainbow trout 94.1 92.1 86.7 79.0 Rainbow trout 78.4 77.7 71.6 65.2 Rainbow trout 83.1 77.2 67.7 57.8 Skamania steelhead 73.6 49.6 28.7 28.0 Ganaraska steelhead 79.1 72.6 73.5 3.5 ix. Conclusions: The data in this study support safety of hydrogen peroxide applied to rainbow trout eggs at a concentration of 566 mg/L. 3. Target Animal Safety Study PMF 005-639: a. Title: “Hydrogen Peroxide Treatment Toxicity to Rainbow Trout Eggs. Part II: Identification of Sensitive Periods During Incubation of Rainbow Trout Eggs.” b. Investigator: Mark P. Gaikowski Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To assess the sensitivity of rainbow trout embryos to hydrogen peroxide by exposing rainbow trout eggs to hydrogen peroxide treatments at concentrations of 0, 566, 1,132, and 3,396 mg/L for 15 minutes daily (Monday through Friday) from 1 to 3 days post-fertilization (? 20 DTU?C) through hatch. ii. Test Animals: Non-eyed, 36 hours old, fertilized rainbow trout (Oncorhynchus mykiss) eggs were allotted in groups of 30 mL to 4 treatment groups containing 3 replicates for each species. Three lots of eggs were used. Treatments were initiated when the eggs were 1 to 3 days old and were performed for 15 minutes once daily (Monday through Friday) until all viable eggs had hatched. iii. Controls: Untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a continuous-flow system. vi. Study Duration: 179 days. vii. Parameters Studied: Percent mortality, temperature, dissolved oxygen, pH, and hydrogen peroxide concentration. viii. Results: Daily percent mortality of rainbow trout eggs increased between Days 6 and 10 (78-135 DTUºC). An increase was also noted between Days 21 and 27. A dose related increase in mortality was also observed, as well as a significant variability by egg lot. ix. Conclusions: There is a period of time during development, Day 6 through Day 10 (78-135 DTUºC), in which rainbow trout embryos may be sensitive to treatments with hydrogen peroxide within the recommended dosage range. This period of time coincides with the closure of the blastopore during embryonic development. 4. Target Animal Safety Study INAD 010-023: a. Title: “Safety of Hydrogen Peroxide to Non-Eyed and Eyed Rainbow Trout (Oncorhynchus mykiss) Eggs.” b. Investigator: Jeffery J. Rach Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To assess the safety of 15 or 45 minute hydrogen peroxide treatments of 0, 1,130, 3,390, 5,650, or 11,300 mg/L administered every other day to non-eyed or eyed rainbow trout (Oncorhynchus mykiss) eggs until eye-up (non-eyed eggs) or until hatch was complete (eyed eggs). ii. Test Animals: Non-eyed, 36 hours old, fertilized rainbow trout (Oncorhynchus mykiss) eggs were allotted in groups of 30 mL to 5 treatment groups containing 3 replicates for each species and for each (non-eyed and eyed) trial. Two lots of eggs were used. Treatments were initiated immediately (non-eyed trial) or after eye-up (eyed egg trial). iii. Controls: Untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a continuous flow system. vi. Study Duration: 23 days. vii. Parameters Studied: Number of live and dead eggs (non-eyed egg trial), number of hatched fry and dead eggs (eyed egg trial), temperature, dissolved oxygen, pH, and hydrogen peroxide concentration. viii. Results: The probability of eye-up was significantly less in the treated non-eyed eggs than in the control eggs (p<0.01). Among treated non-eyed eggs, the probability of egg eye-up was significantly less as treatment concentration and duration increased. The probability of hatch was significantly less in control group eggs than in treated eyed eggs (p<0.01). Among treated eyed eggs, no significant differences were observed as the treatment concentration increased (p>0.10). Survival of non-eyed eggs to eye-up is illustrated in Table 10, and survival of eyed eggs to hatch is illustrated in Table III.B.4.1 and III.B.4.2: Table III.B.4.1: Survival of Non-Eyed Eggs to Eye-Up Incubation Temperature (±2ºC) Exposure Duration (min) Egg Lot # Treatment Concentration (mg/L) 0 1,130 3,390 5,650 1,1300 % Mean (triplicates) eye-up (±SD) 12 15 9403 96 a (1.59) 86 b* (1.56) 54 c* (19.18) 59 c* (5.09) 42 d (9.62) 12 45 9403 96 a (1.59) 48 b* (10.51) 16 c* (4.54) 4 d* (1.31) NT 15 15 9452 76 a (8.81) 57 b* (2.04) 39 c* (4.35) 33 c* (1.64) 10 d (3.06) 15 45 9452 76 a (8.81) 26 b* (12.25) 10 c* (0.87) 5 c* (0.96) NT NT = concentration not tested Within a given experimental design (same treatment duration or incubation temperature), means with the same letter are not significantly different (P? 0.10). Between exposure durations for a given egg lot, means of a given concentration with an asterisk (*) are significantly different (P? 0.10). Table III.B.4.2: Survival of Eyed Eggs to Hatch Incubation Temperature (±2ºC) Exposure Duration (min) Egg Lot # Treatment Concentration (mg/L) 0 1,130 3,390 5,650 11,300 % Mean (triplicates) hatch (±SD) 12 15 9403 90 a (4.65) 97 b (1.42) 95 b (0.71) 97 b (1.06) 97 b (1.84) 12 45 9403 90 a (4.65) 95 b (1.83) 92 b (4.31) 95 b (1.31) NT 15 15 9452 90 a (2.26) 88 a (4.38) 88 a (1.99) 89 a (1.78) 85 a (1.66) 15 45 9452 90 a (2.26) 86 a (2.23) 85 a (2.85) 84 a (2.93) NT NT = concentration not tested Within a given experimental design (same treatment duration or incubation temperature), means with the same letter are not significantly different (P? 0.10). ix. Conclusions: Hydrogen peroxide has no adverse effects on the hatchability of eyed eggs when applied every other day at concentrations as high as 10 times the recommended concentration for the intended therapeutic duration (15 minutes). Additionally, the drug was found not to be harmful to trout egg development when applied every other day for 3 times the recommended duration for up to 5 times the recommended concentration. Hydrogen peroxide has detrimental effects on non-eyed eggs. 5. Target Animal Safety Study INAD 010-023: a. Title: “Safety of hydrogen peroxide to paddlefish Polyodon spathula and rainbow trout Oncorhynchus mykiss eggs” b. Investigator: Jeffrey J. Rach, et al Upper Midwest Environmental Sciences Center 2630 Fanta Reed Road La Crosse, WI 54603 c. General Design: i. Purpose: To determine the safety (measured by toxicity) of daily 15 min hydrogen peroxide treatments of 1,000, 1,500, 2,000, or 2,500 mg/L administered to paddlefish (Polyodon spathula) and rainbow trout (Oncorhynchus mykiss) eggs until hatch was complete. ii. Test Animals: Paddlefish and rainbow trout non-eyed eggs were received within 36 hours of spawning. Eggs were allocated in 30 mL volumes to control groups and treatment groups receiving 1,000, 1,500, 2,000, and 2,500 mg/L. The paddlefish eggs were tested with three replicates of each concentration. Rainbow trout eggs were tested in 2 banks, with three replicates of each concentration per bank. Each group was treated for 15 minutes once daily, until all viable eggs had hatched. iii. Controls: Untreated eggs. iv. Dosage Form: Hydrogen peroxide (35% w/w). v. Route of Administration: Immersion in a continuous-flow system. vi. Study Duration: 8 days for paddlefish, 27 days for rainbow trout. vii. Parameters Studied: Mean percent hatch, water flow, temperature, dissolved oxygen, pH, water hardness, hydrogen peroxide concentration. viii. Results: Refer to Table III.B.5: Table III.B.5: Mean Percent Hatch by Treatment Concentration of H2O2 (mg/L) Species 0 1,000 1,500 2,000 2,500 Rainbow trout 75.7a 52.2b 47.5bc 44.0c 34.5d Paddlefish 7a 35b 26b 27b 25b abcd Means with different superscripts differ (p<.10) ix. Conclusions: Hydrogen peroxide treatments of 1,000 to 2,500 mg/L significantly reduced rainbow trout egg hatch in comparison to the untreated control group. The higher percent egg hatch in the controls (compared to the treated eggs) supports previous studies that determined salmonid eggs are sensitive to hydrogen peroxide concentrations exceeding 500 mg/L when exposed before the closure of the blastopore (78-135 DTU’s °C) during egg maturation. After this sensitive period, daily hydrogen peroxide treatment concentrations could be increased up to 1,000 mg/L. Paddlefish eggs can be treated daily with hydrogen peroxide concentrations up to 1,000 mg/L for 15 min. Hatchery personnel should be advised to conduct a preliminary bioassay on a small number of eggs before treatment of a large batch of eggs since egg sensitivity can vary between species, strains, and unique environmental conditions encountered at each fish culture facility. HYDROGEN PEROXIDE (35 % W/W) TABLE OF CONTENTS PMF 005-639 Page i HYDROGEN PEROXIDE (35 % W/W) GENERAL INFORMATION PMF 005-639 Page 2 HYDROGEN PEROXIDE (35 % W/W) EFFECTIVENESS HYDROGEN PEROXIDE (35 % W/W) APPROVED LABELING