North American Journal of Aquaculture 68:276–280, 2006 Ó Copyright by the American Fisheries Society 2006 DOI: 10.1577/A05-037.1
[Communication]
Reduced Formalin and Hydrogen Peroxide Treatments during Walleye Egg Incubation CRAIG A. SOUPIR* South Dakota Department of Game, Fish, and Parks, Region IV District Office, 603 East 8th Avenue, Webster, South Dakota 57274, USA
MICHAEL E. BARNES South Dakota Department of Game, Fish, and Parks, McNenny State Fish Hatchery, 19619 Trout Loop, Spearfish, South Dakota 57783, USA Abstract.—Formalin use was evaluated as part of an investigational new animal drug study to determine its effectiveness in controlling fungus (water molds: Saprolegniaceae) on incubating eggs of the walleye Sander vitreus. Hydrogen peroxide was also evaluated as an egg treatment alternative to formalin. In total, three antifungal chemical treatment regimes (15 min daily) were evaluated during this study. Eggs were treated with 200 mg of hydrogen peroxide/L of water, 834 mg formalin/L, and the industry standard of 1,667 mg formalin/L. Untreated control eggs were severely infested with fungus and experienced survival to hatch that was significantly lower than that of the chemically treated eggs. Formalin and hydrogen peroxide treatments both controlled fungus and produced similar egg survival to initial hatch. However, walleye egg survival was significantly greater in jars treated with 1,667 mg formalin/L than in any other treatment. This study provided important new data on the use of chemical treatment concentrations lower than the standard of 1,667 mg formalin/L or 500 mg hydrogen peroxide/L to control fungus on incubating walleye eggs.
Fungi (water molds) of the family Saprolegniaceae (e.g., Saprolegnia diclina, Achlya hoferi, and Dictyuchus spp.) are ubiquitously distributed in freshwater and are an important consideration in fish culture (Piper et al. 1982). Egg incubation of walleyes Sander vitreus provides ideal conditions for fungal infection due to the high loading densities, limited space among individuals, and relatively low water flow through culture units (Gaikowski et al. 2003). Prophylactic chemical treatments with 1,667 mg of formalin/L of water or 500 mg hydrogen peroxide/L are typically used to reduce fungal infestation on incubating walleye eggs (Marking et al. 1994; Gaikowski et al. 2003). Currently, formalin is the only U.S. Food and Drug Administration (FDA) antifungal chemical approved for use on walleye eggs (FDA 2003). Hydrogen peroxide is listed as a ‘‘low regulatory priority’’ fishery * Corresponding author:
[email protected] Received March 21, 2005; accepted October 30, 2005 Published online June 15, 2006
chemical and can also be used at concentrations up to 500 mg/L (Winton 2001). There have been very few studies examining antifungal chemical use on eggs from walleyes or other nonsalmonids. Gaikowski et al. (2003) noted that formalin treatments of 1,667 mg/L for 15 min yielded slightly higher walleye egg hatching percentages than did 15-min, 500-mg hydrogen peroxide/L treatments. They also examined hydrogen peroxide treatments of 283 mg/L every other day in experimental (nonproduction) incubators. To the best of our knowledge, there has been no published information concerning the effectiveness of reduced formalin concentrations during walleye egg incubation or of hydrogen peroxide concentrations less than 500 mg/L in production-scale incubator jar loadings. Because of increasing restrictions on formalin discharge into hatchery effluents (Masters 2004) and the possible negative human health issues associated with formalin use (Marking et al. 1994), information is needed to determine whether reductions from the standard formalin concentration of 1,667 mg/L (Rach et al. 1997) can occur. Reductions in formalin or hydrogen peroxide use would also decrease walleye egg incubation costs. The objective of this study was to compare the effects of a reduced formalin treatment concentration (834 mg/L), a reduced hydrogen peroxide concentration (200 mg/L), and a typical formalin concentration (1,667 mg/L) on walleye egg hatching percentages. Methods All walleye eggs were obtained from the Grand, Moreau, or Cheyenne River (Foster Bay) embayments of Lake Oahe, South Dakota, during April 1995–1998. Spawning crews used the ‘‘dry’’ method of fertilization in which sperm and egg are mixed together before activation with water (Piper et al. 1982). Fertilized eggs were then mixed in a solution of either diatomaceous earth or fuller’s earth to reduce natural egg adhesion (material used to remove egg adhesion was similar within lots). Eggs were rinsed, water hardened (2–4 h),
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and transported to Blue Dog State Fish Hatchery, rural Waubay, South Dakota, for incubation (4 h). Upon arrival at the hatchery, a total of 250,000 eggs (2,000 mL) were measured by volume into McDonald egg incubation jars (14-cm diameter 3 38-cm length) by use of a 500-mL graduated beaker. A fine-screened cover was placed over each of the experimental jars to prevent egg escapement. The incubator comprised a headbox, McDonald egg hatching jars, and a fry capture tank. Well water (9.5–11.78C; total hardness as CaCO3, 317 mg/L; alkalinity as CaCO3, 286 mg/L; pH, 7.7; total dissolved solids, 402 mg/L) entered the headbox and flowed by gravity to the incubation jars and finally to the fry capture tanks. Fifteen-minute flow-through treatments with formalin (Parasite-S; solution of 37% formaldehyde, 6–13% methanol; Western Chemical, Inc., Ferndale, Washington) or hydrogen peroxide (Perox-Aid; 35% by weight; Eka Chemicals, Inc., Marietta, Georgia) were used during this study. Formalin use was evaluated under an FDA investigational new animal drug permit (10–023). All treatments began within 24 h of loading and continued daily until the onset of fry hatch to avoid exposure of walleye fry to formalin. Formalin and hydrogen peroxide concentrations reported in this study were nominal values, and no verification of concentration was conducted. However, Gaikowski et al. (2003) reported that expected and actual chemical concentrations in the rearing units were similar at Blue Dog Hatchery. To determine egg survival to first hatch, we enumerated the number of viable and nonviable eggs in each incubation jar. A total of three replicate samples (.300 eggs/sample) were randomly removed from each incubation jar by means of a rigid tube (roughly 9.5 mm in diameter). The sample tube was slowly inserted into the jar at a random location from the top of the jar to bottom, the exposed end of the tube was sealed with a thumb, and the tube containing the eggs was removed. Eggs were placed onto a counting wheel, and the number of viable and nonviable eggs was determined by inspection under a dissecting microscope (33 magnification). Egg survival was estimated as the number of viable eggs divided by the total number of eggs counted. Percent hatch could not be used as a measure of egg survival because walleye fry incubated in individual jars were collected into combined collection tanks. Eggs were visually examined throughout egg incubation, and any presence of fungus was noted. Two replicate jars comprised an experimental treatment (N ¼ 2), which was repeated with four separate egg lots (eggs derived from a single location on a given day) during each year of this study. Therefore, a total of eight egg jars were included in
each treatment, and a single egg jar was the experimental unit (Table 1). Differences in spawning dates, hatchery functions, and personnel resulted in minor differences in methodology during the 4-year study. Otherwise the methods used were similar among years. 1995 procedures.—Eggs were collected between April 26 and 27 from the Grand and Moreau River embayments. Treatments consisted of a negative control (no chemical treatment), 834 mg formalin/L, or 200 mg hydrogen peroxide/L. Treatments began on April 27 and continued daily through May 8 (12 d). 1996 procedures.—Eggs were collected between April 30 and May 1 from the Grand and Moreau River embayments. The trial was the same in design as the 1995 trial; treatments included a negative control (no chemical treatment), 834 mg formalin/L, and 200 mg hydrogen peroxide/L. Treatments began on May 1 and continued daily through May 11 (11 d). Immediately upon commencement of hatching, dead eggs were siphoned from each egg incubation jar and were examined visually for the presence of fungus. 1997 procedures.—Eggs were collected on May 3 and 4 from the Grand and Moreau River embayments. Instead of a negative control (no chemical treatment), a positive control (1,667 mg formalin/L) was compared with 834-mg formalin/L and 200-mg hydrogen peroxide/L treatments. Treatments began on May 4 and continued daily through May 16 (11 d). 1998 procedures.—Eggs were collected between April 30 and May 2 from the Foster and Moreau River embayments. Only 834- and 1,667-mg formalin/L treatments were included in this experiment. Treatments began on May 1 and continued daily through May 9 (9 d). Data were analyzed by analysis of variance with SYSTAT version 10.2 for Windows (SYSTAT 2002). Percentage data were arcsine transformed before analysis to control variance (Steel et al. 1997). Differences in means between treatments were evaluTABLE 1.—Experimental design utilized during a study of hatch percentages for walleye eggs treated with hydrogen peroxide or formalin to control fungal infestation during 1995–1998. The experimental unit was an individual egg jar, which was replicated two times for each of four (repeated) egg lots (eggs derived from a single spawning location on a given day). This design was repeated for each experimental treatment within years; each year’s experiment was independent. Experimental treatment Lot 1 Jar 1
Jar 2
Lot 2 Jar 1
Jar 2
Lot 3 Jar 1
Jar 2
Lot 4 Jar 1
Jar 2
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ated with Fisher’s protected least-significant-difference multiple comparison test; P-values less than or equal to 0.05 were considered to indicate significance. Results and Discussion During the years in which a negative control (no treatment) was included, the percent survival of walleye eggs to hatch in the control jars was significantly lower than that of eggs treated with formalin or hydrogen peroxide (Tables 2, 3). Relative to the untreated controls, over 30% more walleye eggs treated with either chemical survived to first hatch in 1995. While not as dramatic, 13% more walleye eggs hatched in 1996 in the chemically treated jars than in control jars. Untreated control jars exhibited extreme fungal growth by the fifth incubation day in each of the 2 years they were included in the study, which likely contributed to egg mortality rates greater than 50%. Because of shortages in the number of eggs available for experimentation and the obvious need for antifungal chemical treatments, the negative control was not used in the last 2 years of the study. Of the incubation jars treated with reduced formalin or hydrogen peroxide in 1995 and 1996, only two jars demonstrated fungal growth, which was minimal and typically limited to a few small egg masses. Fungal development in treated incubation jars was not detected until day 11 of incubation during any year. The lack of fungal growth, particularly in the 200-mg hydrogen peroxide/L jars, is somewhat surprising given the fungal infestations on walleye eggs treated with 500 mg hydrogen peroxide/L as reported by Gaikowski et al. (2003) and the fungal growth reported on salmonid eggs receiving hydrogen peroxide concentrations of 500 mg/L or greater (Waterstrat and Marking 1995; Barnes et al. 1998; Arndt et al. 2001; Barnes and Gaikowski 2004). Significantly greater egg survival to hatch was observed for the normative 1,667-mg formalin/L treatment than for the 834-mg formalin/L and 200mg hydrogen peroxide/L treatments. Overall, the mean rate of survival was 7% higher when 1,667 mg formalin/L was used (Figure 1). Because of low statistical power, the small sample sizes (N ¼ 2) used within each lot in this study probably precluded statistical significance (Curtis et al. 1991). However, mean survival within each lot was consistently, though nonsignificantly, greater in the jars receiving formalin at 1,667 mg/L than in any of the other treatments, further strengthening the statistically significant increase in survival observed when all lots were combined. Considering that a 7% increase in fry survival during a normal production year at the Blue Dog Hatchery would result in the production of approximately 4,000,000 more walleye fry (Broughton
TABLE 2.—Analysis of variance results indicating effect, degrees of freedom (df), F-ratio, and probability of committing a type I error (P) in a study of hatch percentages of walleye eggs that received no chemical treatment (negative control) or that were treated with hydrogen peroxide (200 mg/ L [H200]) or formalin (834 mg/L [F834] or 1,667 mg/L [F1667; positive control]) to control fungal infestation. Percentage data were arcsine-transformed before analysis to control variance. Differences in means between treatments were evaluated with Fisher’s protected least-significantdifference multiple comparison test when P was 0.05. Main effect
df
F-ratio
P
1995 (No treatment, F834, H200) 3 7.8 2 13.4 2 24.2 2 10.3 2 12.5 2 58.7 12 1.3
0.004 0.032 0.014 0.046 0.035 ,0.001 0.312
1996 (No treatment, F834, H200) Lot 3 20.3 1 2 ,0.1 2 2 18.0 3 2 622.6 4 2 7.3 Treatment 2 128.9 Lots 3 treatment 6 7.2
,0.001 0.007 0.021 ,0.001 0.070 ,0.001 0.002
Lot 1 2 3 4 Treatment Lot 3 treatment
Lot 1 2 3 4 Treatment Lots 3 treatment
1997 (F834, H200, F1667) 3 7.7 2 4.4 2 6.6 2 36.4 2 12.0 2 23.8 6 3.4
0.004 0.129 0.079 0.008 0.037 ,0.001 0.035
Lot 1 2 3 4 Treatment Lot 3 treatment
1998 (F834, H200, F1667) 3 14.7 1 4.9 1 0.6 1 46.7 1 3.9 1 23.9 3 3.4
0.001 0.158 0.510 0.021 0.188 0.001 0.075
et al. 2004), formalin treatments of 1,667 mg/L would produce tremendous increases in rearing efficiency, including both the costs and labor associated with egg collection. The hydrogen peroxide and 834-mg formalin/L treatments produced similar egg survival percentages. However, there was considerable variation in some of the jars treated with hydrogen peroxide. In particular, large standard errors occurred with hydrogen peroxide treatments in two of the four lots during 1997. A possible explanation for this variability is that egg quality, as indicated by overall survival, decreased dramatically from 1995 to 1997. Differences in egg quality have been shown to influence the effect of 1,667-mg formalin/L treatments on salmonid egg survival (Barnes et al. 2000). It is conceivable that
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TABLE 3.—Mean (SE) percent survival to first hatch, estimated indirectly from egg viability, of walleye eggs that received either no chemical treatment (control), daily 15-min formalin treatments (834 or 1,667 mg/L), or daily 15-min hydrogen peroxide treatments (200 mg/L) in 1 of 4 years (1995–1998). Numbers in columns within lots are means of two replicate incubation jars (N ¼ 8 for combined means). Means within a column and within lots (or combined lots) with different letters are significantly different (P 0.05). Year Treatment Lot 1 No treatment (control) Hydrogen peroxide Formalin (834 mg/L) Formalin (1,667 mg/L) Lot 2 No treatment Hydrogen peroxide Formalin (834 mg/L) Formalin (1,667 mg/L) Lot 3 No treatment Hydrogen peroxide Formalin (834 mg/L) Formalin (1,667 mg/L) Lot 4 No treatment Hydrogen peroxide Formalin (834 mg/L) Formalin (1,667 mg/L) Combined lots No treatment (control) Hydrogen peroxide Formalin (834 mg/L) Formalin (1,667 mg/L)
1995
1996
42.5 (7.5) z 70.0 (0.0) y 73.7 (3.8) y
46.6 (2.5) z 64.7 (0.0) y 63.4 (1.3) y
38.7 (8.7) z 80.0 (0.0) y 86.2 (3.8) y
36.2 (2.6) z 56.9 (3.9) y 58.2 (1.3) y
67.5 (2.5) y 83.7 (3.7) z 87.5 (2.5) z
44.0 (0.0) y 71.7 (0.7) z 67.9 (0.7) z
50.0 (7.5) y 81.2 (1.2) y 85.0 (5.0) z
53.6 (0.7 y) 59.5 (0.0) y 62.1 (2.6) y
49.7 (4.9) y 78.7 (2.1) z 83.1 (2.5) z
45.1 (2.5) y 63.2 (2.3) z 62.9 (1.4) z
poor egg quality and the associated increase in the number of easily infested dead eggs (Smith et al. 1986) would have made treatment with 200 mg hydrogen peroxide/L much less effective. It is also possible that differences between spawning locations played a part in the increased variability observed in 1997. The egg lots used in this study came from three different locations of Lake Oahe that have historically differed in survival to hatch during hatchery incubation (Mauk and Brown 2001). In addition to the obvious control of fungus infestations and possible live-egg infections, both the formalin and hydrogen peroxide treatments may have benefited the eggs by reducing bacterial loadings (Stephenson et al. 2003; Barnes et al. 2005). Decreased bacterial numbers may also help explain the greater survival in the eggs receiving 1,667 mg formalin/L than in eggs receiving either 834 mg formalin/L or 200 mg hydrogen peroxide/L. The benefits of daily 15-min flow-through treatments of incubating walleye eggs with 1,667 mg formalin/L are apparent. However, consideration should be given to the human health (Marking et al. 1994) and environmental issues (Masters 2004) associated with formalin use. Reduced formalin con-
1997
1998
46.2 (1.2) y 51.9 (3.1) y 54.4 (0.6) y
48.7 (1.3) y 55.0 (2.5) y
34.4 (9.4) y 52.5 (0.0) y 60.0 (0.0) y
57.5 (2.5) y 60.7 (3.3) y
45.6 (0.6) y 45.0 (0.0) y 50.6 (0.6) z
54.2 (1.8) y 68.7 (1.2) z
46.9 (6.9) z 56.9 (0.6) yz 71.9 (0.6) y
63.0 (2.0) y 67.0 (0.5) y
43.3 (2.9) y 51.6 (1.7) y 59.2 (3.0) z
55.9 (2.1) y 62.9 (2.2) z
FIGURE 1.—Mean percent survival, estimated indirectly from egg viability, of walleye eggs in an 834-mg formalin/L treatment (15 min daily) relative to the survival of eggs treated with 1,667 mg formalin/L or 200 mg hydrogen peroxide/L at Blue Dog State Fish Hatchery, Waubay, South Dakota (1995– 1998). Deviations from the diagonal line represent percentage differences in mean percent survival between the 834-mg formalin/L treatment and the other treatments.
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centrations or very low (200-mg/L) concentrations of hydrogen peroxide may be of use if egg supplies are not unduly limited, reductions on the effluent discharge of formalin are required, or limiting the formalin exposure of hatchery staff is desired. Future studies should be conducted to determine whether a reduction in treatment duration, concentration, or interval could provide an effective alternative to daily treatments with 1,667 mg formalin/L. Acknowledgments We thank Clark Moen, Jerry Broughton, Randy Smidt, Eugene Holm, and Robert Whitlock of the South Dakota Department of Game, Fish, and Parks. References Arndt, R. E., E. J. Wagner, and M. D. Routledge. 2001. Reducing or withholding hydrogen peroxide treatment during a critical stage of rainbow trout development: effects on eyed eggs, hatch, deformities, and fungal control. North American Journal of Aquaculture 63:161– 166. Barnes, M. E., D. E. Ewing, R. J. Cordes, and G. L. Young. 1998. Observations on hydrogen peroxide control of Saprolegnia spp. during rainbow trout egg incubation. Progressive Fish-Culturist 60:67–70. Barnes, M. E., K. Wintersteen, W. A. Sayler, and R. J. Cordes. 2000. Use of formalin during incubation of eyed rainbow trout eggs. North American Journal of Aquaculture 62:54–59. Barnes, M. E., and M. P. Gaikowski. 2004. Use of hydrogen peroxide during incubation of landlocked fall Chinook salmon eggs in vertical-flow incubators. North American Journal of Aquaculture 66:29–34. Barnes, M. E., D. Bergmann, H. Stephenson, M. Gabel, and R. J. Cordes. 2005. Bacterial numbers from landlocked fall Chinook salmon eyed eggs subjected to various formalin treatments as determined by scanning electron microscopy and bacteriological culture methods. North American Journal of Aquaculture 67:23–33. Broughton, J., R. Smidt, C. Soupir, E. Holm, and R. Whitlock. 2004. Blue Dog State Fish Hatchery 2003 annual production report. South Dakota Department of Game, Fish, and Parks, Annual Report 04–02, Pierre. Curtis, C. R., M. D. Salman, and S. Shott. 1991. Power and sample size. Journal of the American Veterinary Medical Association 197:838–840.
FDA (U.S. Food and Drug Administration). 2003. Drugs approved for use in aquaculture (poikilothermic food species). Available: http://www.fda.gov/cvm/index/ aquaculture/aqualibtoc.htm#ApprovedDrugs. (October 2003). Gaikowski, M. P., J. J. Rach, M. Drobish, J. Hamilton, T. Harder, L. A. Lee, C. Moen, and A. Moore. 2003. Efficacy of hydrogen peroxide in controlling mortality associated with saprolegniasis on walleye, white sucker, and paddlefish eggs. North American Journal of Aquaculture 65:349–355. Mauk, R. J., and M. L. Brown. 2001. Performance of walleye progeny from Missouri River tributaries in South Dakota. North American Journal of Aquaculture 63:167–170. Marking, L. L., J. J. Rach, and T. M. Schreier. 1994. Evaluation of antifungal agents for fish culture. Progressive Fish-Culturist 56:225–231. Masters, A. L. 2004. A review of methods for detoxification and neutralization of formalin in water. North American Journal of Aquaculture 66:325–333. Piper, R. G., I. B. McElwain, L. E. Orme, J. P. McCraren, L. G. Fowler, and J. R. Leonard. 1982. Fish hatchery management. U.S. Fish and Wildlife Service, Washington, D.C. Rach, J. J., G. E. Howe, and T. M. Schreier. 1997. Safety of formalin treatments of warm- and coolwater eggs. Aquaculture 149:183–191. Smith, S. N., R. A. Armstrong, J. Springate, and G. Barker. 1986. Infection and colonization of trout eggs by Saprolegniaceae. Transactions of the British Mycological Society 85:719–764. Steel, R. G. D., J. H. Torrie, and D. A. Dickey. 1997. Principles and procedures of statistics: a biometrical approach, 3rd edition. McGraw-Hill, New York. Stephenson, H., M. Gabel, and M. E. Barnes. 2005. Microbial inhibition in response to treatments of hydrogen peroxide and formalin on landlocked fall Chinook salmon eyed eggs, as determined by scanning electron microscopy. North American Journal of Aquaculture 65:324–329. SYSTAT. 2002. SYSTAT for Windows, version 10.2. SYSTAT, Evanston, Illinois. Waterstrat, P. R., and L. L. Marking. 1995. Clinical evaluation of formalin, hydrogen peroxide, and sodium chloride for the treatment of Saprolegnia parasitica on fall Chinook salmon eggs. Progressive Fish-Culturist 57:287–291. Winton, J. R. 2001. Fish health management. Pages 559–640 in G. Wedemeyer, editor. Fish hatchery management, 2nd edition. American Fisheries Society, Bethesda, Maryland.
