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

2. 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

3. 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.

4. 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

5. 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

6. 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.

7. 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

8. 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

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

10. Blind Pony SFH - paddlefish
Temperature °C
Four trials - one or two females per trial
500, 750, or 1000 mg/L
% hatch ranged from % (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%

11. 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.

12. 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%

13. 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; %
Some small fungus clumps observed in hydrogen peroxide treatment jars, no fungus in formalin jars.

14. 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.

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

16. 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% % %

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

18. 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

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

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

21. Rathbun SFH - channel catfish eggs
supporting trial
disease identification pending
concentration verification
all concentrations were 50-60% of target
500 mg/l was
750 mg/L was
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

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

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

24. 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.