Laboratory and Production Scale Disinfection of Salmonid Eggs with Hydrogen Peroxide

Disinfection tests were conducted on eggs from rainbow trout Oncorhynchus mykiss, cutthroat trout O. clarkii, and brown trout Salmo trutta to evaluate hydrogen peroxide (H2O2) as an egg disinfectant. A daily drip of 500 mg/L hydrogen peroxide for 35 min on eyed brown trout eggs safely led to significantly reduced bacterial abundance relative to untreated controls, but abundance did not differ significantly from that in a formalin treatment (2,000 mg/L for 15 min). Using water-hardened cutthroat trout eggs, hydrogen peroxide concentrations of (1) 10 g/L for 2 min, (2) 10 g/L for 3 min, (3) 15 g/L for 2 min, and (4) 1,000 mg/L for 15 min were compared with two controls (untreated; 100 mg/L iodine). Bacteria were significantly more abundant in the untreated eggs, but abundance did not significantly differ among chemical treatments. In another test, rainbow trout eggs treated the day before hatch with 15 g/L H2O2 for 2 min had significantly higher mortality than the controls. In production-scale tests, 10–15 g/L H2O2 for 2 min was safe for rainbow and cutthroat trout eggs and significantly reduced bacterial abundance relative to that in untreated eggs. Complete disinfection was not achievable with either iodine or hydrogen peroxide. Daily drip treatment with either formalin or hydrogen peroxide significantly reduced bacterial growth and is recommended. Fungal and bacterial growth on fish eggs can seriously compromise egg survival during incubation (Ross and Smith 1972; Barnes et al. 2003), owing in part to egg membrane degradation (Pavlov and Moksness 1993; Morrison et al. 1999; Barnes et al. 2009). Since the use of malachite green was discontinued because of human health concerns, formalin and iodine have been used routinely in the last few decades to control fungal and bacterial growth on fish eggs (Amend 1974; Barnes et al. 1997; Barnes et al. 1998). However, current problems with vertically transmitted bacteria such as Flavobacterium psychrophilum (the causative agent of bacterial coldwater disease, also known as rainbow trout fry syndrome) and with microsporidiosis (e.g., Loma salmonae) indicate that current disinfection protocols need revision (Kumagai et al. 1998; Shaw et al. 1999; Wagner et al. 2008; Barnes et al. 2009). Furthermore, Presterl et al. (2007) observed that bacterial biofilms treated with stock concentrations of povidone iodine (1% active iodine) were still viable after a 30 min exposure. *Corresponding author: [email protected] Received March 9, 2011; accepted May 14, 2011 There has been some research evaluating alternatives to the traditional iodineand formalin-based disinfection (Bailey and Jeffrey 1989; Schrader 2008; Copur et al. 2010). Hydrogen peroxide (H2O2) has emerged as an alternative that is both effective and environmentally benign, breaking down into hydrogen, oxygen, and water (Marking et al. 1994). Hydrogen peroxide has controlled fungus when applied in daily prophylactic treatments (Gaikowski et al. 1998; Arndt et al. 2001). Treatments of 0.5% to 1.0% for 15–60 min controlled fungal growth in the eggs of rainbow trout Oncorhynchus mykiss (Schreier et al. 1996; Barnes et al. 1998). The bacteriological effects of treating eggs with hydrogen peroxide are less studied. Recent data, however, indicate that 30 g/L H2O2 for 5 min controls or significantly reduces bacterial growth on the eggs of red drum Sciaenops ocellatus (Douillet and Holt 1994), Atlantic cod Gadus morhua (Peck et al. 2004), and haddock Melanogrammus aeglefinus (Peck et al. 2004). Rainbow trout eggs treated with 0.5–2.0 g/L H2O2 for 15 min 92 D ow nl oa de d by [ E ri c J. W ag ne r] a t 0 9: 54 0 4 Fe br ua ry 2 01 2 DISINFECTION OF SALMONID EGGS WITH HYDROGEN PEROXIDE 93 had significantly reduced bacterial abundance, but a 100 mg/L iodine treatment provided better control of bacteria (Wagner et al. 2008). Wagner et al. (2010) found that 30 g/L H2O2 for 1 min or 15 g/L for 2 min significantly reduced bacterial loads on eggs without compromising rainbow trout egg survival. Effects of these concentrations on cutthroat trout Oncorhynchus clarkii or brown trout Salmo trutta have not been tested. Productionscale tests are also needed. Research on formalin has demonstrated that it has improved survival of eggs and controls fungal growth (Watanabe 1940; Barnes et al. 1997, 2000; Rach et al. 2005; Small and Chatakondi 2006), but the bactericidal ability of formalin has not been studied adequately. Wright and Snow (1975) observed that formalin concentrations of up to 2,000 mg/L for 15 min were insufficient to kill Aeromonas liquefaciens. Wagner et al. (2008) found that eggs exposed to concentrations of 1,000 mg/L formalin or less had similar bacterial abundance as untreated eggs; however, 1,667–2,000 mg/L formalin led to significant reductions in bacteria numbers. In this study, the effects of hydrogen peroxide are summarized for several different experiments. These evaluated the effects of various hydrogen peroxide concentrations on cutthroat trout eggs, the bactericidal effects of hydrogen peroxide daily drip treatments relative to those of formalin, and the effects of production-scale egg treatment with hydrogen peroxide. Problems with treating eggs just prior to hatch with hydrogen peroxide are also documented in this article.