Presentation on theme: "Hydrogen peroxide INAD (Perox-AidTM)"— Presentation transcript:
1Hydrogen peroxide INAD 10-023 (Perox-AidTM) Efficacy of hydrogen peroxide to control Saprolegniasis on cool- and warmwater fish eggsHydrogen peroxide INAD(Perox-AidTM)
2Egg Efficacy Trial Participants Mark Drobish – Gavins Point NFHLynn Lee – UMESCAndy Moore – Rathbun ResearchClark Moen – Blue Dog SFHTom Harder – Max McGrawDennis Smith – Dundee SFHJerry Hamilton – Blind Pony SFH
3INAD #10-023 Study CAP-00-FUNGUS Study CAP-00-PARASITES submitted to CVM 03 March 2000Twenty-nine participating facilitiesStudy CAP-00-PARASITESsubmitted to CVM 18 April 2000Twenty-one participating facilitiesStudy CAP-00-BACTERIATwenty-three participating facilitiesCollect efficacy data to support the approval of Perox-AidTM (35% H2O2).Provide an opportunity for fish culturists to legally use Perox-AidTM (35% H2O2) to control or prevent external parasites and control or prevent mortalities associated with BGD or external flavobacteriosis on fish.
4INAD #10-023 Participant Locations Western ChemicalsEka Chemicals, IncUMESCINAD holder: UMESCTest Article Source: Eka Chemicals, Inc. Marietta GA; Western Chemical Ferndale WA35% H2O2 (same formulation as the Perox-AidTM distributed in Canada) 55 gal. drum or 5 gal. carboyStudy Monitors: Jeff Rach and Mark Gaikowski
5Protocol Methods Monitor / Record Mortality Identify Disease “Control” Station History“Prevention”Select therapy and treatconcentrationdurationfrequencyrepetitionChemical InventoryControls?Monitor / Record MortalityAdverse Reactions
6Hydrogen peroxide therapy - Fish Duration: 30 or 60 minConcentration50, 75, or 100 mg/L for 60 min100 or 150 mg/L for 30 minRepetition/Interval: once daily on consecutive or alternate days for up to 10 treatments.Conduct most treatments at between 50 or 75 mg/L for 60 min or 100 mg/L for 30 min to show efficacy at the low end of the label claim.Conduct bioassays.Percid and Esocid initial treatments should be <75 mg/L for <60 min.No concomitant therapy.
7Hydrogen peroxide therapy - Eggs Duration: 15 minConcentration: 500 or 750 mg/Lminimum incubator concentration is 500 mg/L for 15 minRepetition/Interval: Once daily on consecutive or alternate days through hatch
8Egg Efficacy Trials Seven egg efficacy trials conducted Blind Pony SFH, Jerry Hamilton, paddlefishBlue Dog SFH, Clark Moen, walleyeDundee SFH, Dennis Smith, smallmouth bassGavins Point NFH, Mark Drobish, walleyeMax McGraw, Tom Harder, walleyeRathbun SFH, Alan Moore, channel catfishUMESC, Lynn Lee, walleyeFungus identified in three trialsFungus identified in three of the seven trials
9Saprolegnia parasitica Gemmae (red arrow)Zoospores in zoosporangium(blue arrow)Photo credit: Steve Carpenter, Ph.D., Abbey Lane Labs.
10Blind Pony SFH - paddlefish Temperature °CFour trials - one or two females per trial500, 750, or 1000 mg/L% hatch ranged from % (of fertile eggs)2 - 4 consecutive treatments; fungus in all treated jarsnoted cessation of egg development after hydrogen peroxide treatmentwithholding treatments until 2 d post fertilization resulted in basically no hatchcompared rolling w/o treatment to hydrogen peroxide treatment w/o rollingrolled eggs - 60% hatch treated eggs 24% hatchNot pivotal trialsNo concentration verification performedPsuedo-replication - same concentration tested in two trials but not simultaneouslyNo controlsMost egg takes were described as “bad” by the Investigator - ie., low fertilityIn a set of exposures conducted by UMESC in 2000, did not see cessation of embryo developmentIn another safety study conducted at UMESC in 2000, percent hatch in PAH eggs treated at 1000 to 2500 ranged from1000 mg/L – 34%1500 mg/L – 26%2000 mg/L – 27%2500 mg/L – 24%In an earlier study conducted by UMESC, had mean percent hatch of0 mg/L – 72%1000 mg/L – 82%3000 mg/L – 53%6000 mg/L – 42%
11Blind Pony - paddlefish Saprolegnia ferax Oogonium with oospores showing cell wall pitting.Oogonia and gemmae from sterile house fly culture.Photo credit: Steve Carpenter, Ph.D., Abbey Lane Labs.
12Max McGraw - walleye ~ 1 million eggs total x = 72% x = 68% Not a pivotal trialfungus not identifiedno concentration verificationInitial egg number generally less in control jars than in treatment jars.Overall % hatchcontrol - 72% treatment - 68%Few fungused eggs in treated jars, control jars were siphoned to limit fungus spreadCouldn’t afford to lose control eggsx = 72%x = 68%
13Blue Dog SFH - walleye ~1.5 million eggs total Pivotal trial ReplicationComparison to approved therapy (formalin at 1667 ppm)concentration verification (+12%)randomization procedures followeddisease not identifiedAlthough treatments were significantly different when compared by lot, the overall difference in % hatch was minimal; %Some small fungus clumps observed in hydrogen peroxide treatment jars, no fungus in formalin jars.
14Gavins Point NFH - walleye ~3.1 million eggs totalPivotal trialReplicationcontrolsconcentration verificationrandomization procedures followedblindingdisease not identifiedSignificant increase in the probability of hatch as concentration increases.Large clumps of fungused eggs observed and removed from controls.
15Gavins Point NFH - walleye 500 mg/LControl750 mg/LPhoto credit: Mark Drobish, Gavins Point NFH
16UMESC - walleye ~2.7 million eggs total Pivotal trial Replication controlsconcentration verificationrandomization procedures followedblindingdisease identified S. parasiticaRelatively small clumps observed in controls - very few clumps in treated eggs% eye-up0 - 74%%%
17UMESC - Walleye Lynn recovering from a late night Study techs - Wendy and SueChelsea Berg not pictured
18Dundee SFH - smallmouth bass eggs ~ 2500 eggs per jar~30,000 eggs totalPivotal trialdisease identified Saprolegnia parasiticareplicationconcentration verificationcontrolsblindingFungus present in all jarsOverall hatch not different
19Dundee SFH - smallmouth bass eggs Number of fungused eggs removed during incubation.Definitely more fungused eggs removed from controls than treatment.
20Dundee SFH - smallmouth bass fry ~ 2500 fry per tank~30,000 fry total
21Rathbun SFH - channel catfish eggs supporting trialdisease identification pendingconcentration verificationall concentrations were 50-60% of target500 mg/l was750 mg/L washad a bad drum of H2O2 – sample verified by Eka Chemicals was 15% not 35%replicationcontrolsFungus present only in control jars – fungal id as S. parasitica
22Rathbun SFH - channel catfish control eggs Photo credit: Andy Moore, Rathbun SFH
24ConclusionsA hydrogen peroxide treatment regimen of 500 mg/L administered for 15 min on consecutive days from incubation to hatch is effective in controlling fungus on walleye and channel catfish eggs.Hydrogen peroxide virtually eliminated fungal growth in both walleye and channel catfish eggs and increased the probability of walleye hatch in two of three controlled trials.