Hydrogen peroxide INAD 10-023 (Perox-AidTM) Efficacy of hydrogen peroxide to control Saprolegniasis on cool- and warmwater fish eggs Hydrogen peroxide INAD 10-023 (Perox-AidTM)

Egg Efficacy Trial Participants Mark Drobish – Gavins Point NFH Lynn Lee – UMESC Andy Moore – Rathbun Research Clark Moen – Blue Dog SFH Tom Harder – Max McGraw Dennis Smith – Dundee SFH Jerry Hamilton – Blind Pony SFH

INAD #10-023 Study CAP-00-FUNGUS Study CAP-00-PARASITES submitted to CVM 03 March 2000 Twenty-nine participating facilities Study CAP-00-PARASITES submitted to CVM 18 April 2000 Twenty-one participating facilities Study CAP-00-BACTERIA Twenty-three participating facilities Collect 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.

INAD #10-023 Participant Locations Western Chemicals Eka Chemicals, Inc UMESC INAD holder: UMESC Test Article Source: Eka Chemicals, Inc. Marietta GA; Western Chemical Ferndale WA 35% H2O2 (same formulation as the Perox-AidTM distributed in Canada) 55 gal. drum or 5 gal. carboy Study Monitors: Jeff Rach and Mark Gaikowski

Protocol Methods Monitor / Record Mortality Identify Disease “Control” Station History “Prevention” Select therapy and treat concentration duration frequency repetition Chemical Inventory Controls? Monitor / Record Mortality Adverse Reactions

Hydrogen peroxide therapy - Fish Duration: 30 or 60 min Concentration 50, 75, or 100 mg/L for 60 min 100 or 150 mg/L for 30 min Repetition/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.

Hydrogen peroxide therapy - Eggs Duration: 15 min Concentration: 500 or 750 mg/L minimum incubator concentration is 500 mg/L for 15 min Repetition/Interval: Once daily on consecutive or alternate days through hatch

Egg Efficacy Trials Seven egg efficacy trials conducted Blind Pony SFH, Jerry Hamilton, paddlefish Blue Dog SFH, Clark Moen, walleye Dundee SFH, Dennis Smith, smallmouth bass Gavins Point NFH, Mark Drobish, walleye Max McGraw, Tom Harder, walleye Rathbun SFH, Alan Moore, channel catfish UMESC, Lynn Lee, walleye Fungus identified in three trials Fungus identified in three of the seven trials

Saprolegnia parasitica Gemmae (red arrow) Zoospores in zoosporangium (blue arrow) Photo credit: Steve Carpenter, Ph.D., Abbey Lane Labs.

Blind Pony SFH - paddlefish Temperature 15 - 16°C Four trials - one or two females per trial 500, 750, or 1000 mg/L % hatch ranged from 14 - 56% (of fertile eggs) 2 - 4 consecutive treatments; fungus in all treated jars noted cessation of egg development after hydrogen peroxide treatment withholding treatments until 2 d post fertilization resulted in basically no hatch compared rolling w/o treatment to hydrogen peroxide treatment w/o rolling rolled eggs - 60% hatch treated eggs 24% hatch Not pivotal trials No concentration verification performed Psuedo-replication - same concentration tested in two trials but not simultaneously No controls Most egg takes were described as “bad” by the Investigator - ie., low fertility In a set of exposures conducted by UMESC in 2000, did not see cessation of embryo development In another safety study conducted at UMESC in 2000, percent hatch in PAH eggs treated at 1000 to 2500 ranged from 1000 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 of 0 mg/L – 72% 1000 mg/L – 82% 3000 mg/L – 53% 6000 mg/L – 42%

Blind 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.

Max McGraw - walleye ~ 1 million eggs total x = 72% x = 68% Not a pivotal trial fungus not identified no concentration verification Initial egg number generally less in control jars than in treatment jars. Overall % hatch control - 72% treatment - 68% Few fungused eggs in treated jars, control jars were siphoned to limit fungus spread Couldn’t afford to lose control eggs x = 72% x = 68%

Blue Dog SFH - walleye ~1.5 million eggs total Pivotal trial Replication Comparison to approved therapy (formalin at 1667 ppm) concentration verification (+12%) randomization procedures followed disease not identified Although treatments were significantly different when compared by lot, the overall difference in % hatch was minimal; 1.9-2.6% Some small fungus clumps observed in hydrogen peroxide treatment jars, no fungus in formalin jars.

Gavins Point NFH - walleye ~3.1 million eggs total Pivotal trial Replication controls concentration verification randomization procedures followed blinding disease not identified Significant increase in the probability of hatch as concentration increases. Large clumps of fungused eggs observed and removed from controls.

Gavins Point NFH - walleye 500 mg/L Control 750 mg/L Photo credit: Mark Drobish, Gavins Point NFH

UMESC - walleye ~2.7 million eggs total Pivotal trial Replication controls concentration verification randomization procedures followed blinding disease identified S. parasitica Relatively small clumps observed in controls - very few clumps in treated eggs % eye-up 0 - 74% 500 - 68% 750 - 71%

UMESC - Walleye Lynn recovering from a late night Study techs - Wendy and Sue Chelsea Berg not pictured

Dundee SFH - smallmouth bass eggs ~ 2500 eggs per jar ~30,000 eggs total Pivotal trial disease identified Saprolegnia parasitica replication concentration verification controls blinding Fungus present in all jars Overall hatch not different

Dundee SFH - smallmouth bass eggs Number of fungused eggs removed during incubation. Definitely more fungused eggs removed from controls than treatment.

Dundee SFH - smallmouth bass fry ~ 2500 fry per tank ~30,000 fry total

Rathbun SFH - channel catfish eggs supporting trial disease identification pending concentration verification all concentrations were 50-60% of target 500 mg/l was 255-272 750 mg/L was306-340 had a bad drum of H2O2 – sample verified by Eka Chemicals was 15% not 35% replication controls Fungus present only in control jars – fungal id as S. parasitica

Rathbun SFH - channel catfish control eggs Photo credit: Andy Moore, Rathbun SFH

Rathbun SFH - channel catfish treated eggs Photo credit: Andy Moore, Rathbun SFH

Conclusions A 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.