Fungicidal Activity of Glutaraldehyde against Causative Agents of Saprolegniasis in Fish Eggs: Saprolegnia parasitica, S. diclina, and S. hypogyna Strains

Abstract

Saprolegniasis causes significant mortality of fish in aquaculture. Historically, malachite green, formaldehyde, and bronopol have been used to control saprolegniasis. However, their use has either been banned or faced issues with unstable supply chains. Given these limitations, there is a pressing need to develop an alternative chemical solution to prevent this disease effectively. This study focused on glutaraldehyde (GA) as a potential chemical and aimed to assess its effectiveness against various Saprolegnia strains. Seven Saprolegnia strains, including S. parasitica, S. diclina, and S. hypogyna were exposed to GA, malachite green, formaldehyde, or bronopol to evaluate their fungicidal effects. The growth of all tested strains was successfully inhibited by GA at concentrations ranging from 53 to 212 mg/L and temperatures between 10 and 20°C, with a minimum exposure duration of 30 min. The high efficacy of GA indicates its potential for controlling saprolegniasis in fish eggs. Additionally, one strain of S. parasitica was exposed to other aldehyde compounds (formaldehyde, glyoxal, acetaldehyde, isobutyraldehyde, malonaldehyde, or valeraldehyde) to assess the effectiveness of GA based on the presence of the aldehyde group. The fungicidal effects of these compounds were observed at higher concentrations (exceeding 405 mg/L) compared to GA, suggesting that the mere presence of aldehyde groups does not necessarily indicate fungicidal action.

Infection by the genus Saprolegnia, belonging to the oomycete group, leads to saprolegniasis, resulting in mass mortality among many fish species in aquaculture. Saprolegnia, commonly known as water molds, is a multicellular organism that assimilates nutrients through extracellular digestion (Bruno et al., 2011). Saprolegnia infections form cotton wool-like structures on eggs or the body surface of fish (Hussein et al., 2001; Liu et al., 2017). These infections can be observed in many fish species at any stage of their life cycle (Pickering and Willoughby, 1982). In adult fish, the invasion of Saprolegnia likely results in impaired osmoregulation (Gardner, 1974; Hargens and Perez, 1975). On eggs, the development of cotton wool-like structures is intensified when dead eggs are not immediately removed (Smith et al., 1984). In aquaculture, saprolegniasis predominantly arises from several species including S. parasitica, S. diclina, and S. hypogyna (van West, 2006; Fregeneda-Grandes et al., 2007; van den Berg et al., 2013; Liu et al., 2017). In salmonid species, infection with S. parasitica is observed in many countries including Northwest Europe, Chile, Japan, and Canada (Hussein and Hatai, 2002; Elameen et al., 2021) and experimental infection with the species causes extremely high mortality in rainbow trout Oncorhynchus mykiss eggs (Kitancharoen and Hatai, 1996). S. diclina infections are associated with fish and amphibian eggs (Diéguez-Uribeondo et al., 2007; Fregeneda-Grandes et al., 2007; Johnson et al., 2008). Moreover, S. hypogyna has been identified in freshwater hatcheries (Fregeneda-Grandes et al., 2007; Liu et al., 2017). Given the challenge of preventing saprolegniasis in fish and fish eggs, it is crucial to utilize chemical treatments.

Previously, malachite green had been used to prevent saprolegniasis (Willoughby and Roberts, 1992). However, malachite green was globally banned in 2002 because of its carcinogenic and hazardous effects (van West, 2006). Since the discontinuation of malachite green, no equally effective treatment has emerged, leading to a resurgence of saprolegniasis (Bruno et al., 2011). Currently, various reagents are used to prevent saprolegniasis. Formalin, a solution of 37% formaldehyde, has been shown to inhibit saprolegniasis in fish and their eggs (Marking et al., 1994; Schreier et al., 1996; Rach et al., 2005; Gieseker et al., 2006). In the United States, the Food and Drug Administration (UFDA) has approved formalin for use in three commercial formulations: Formalin-F™, Formacide-B, and Parasite-S^®; the use is specifically permitted only for salmonid, trout, and esocid eggs (USFDA, 2024). However, formaldehyde has been shown to be carcinogenic to humans based on evidence with the underlying mechanism (NTP, 2021). Therefore, the use of formaldehyde for fish egg disinfection is currently banned in the EU (Laly et al., 2018; Frida, 2021). Additionally, its use is banned in Japan due to legal revisions prohibiting the use of unapproved drugs (Sugiura et al., 2007). Meanwhile, bronopol (2-bromo-2-nitro-1,3-propanediol) is also employed as a preventative measure against saprolegniasis in fish eggs. This chemical preservative is widely utilized across various industries, including medical, pharmaceutical, cosmetic, and personal care products (Bryce et al., 1978; Toler, 1985). It is a potential inhibitor of thiol-containing dehydrogenase enzymes, likely leading to the destruction of cell membranes and subsequent cell death (Shepherd et al., 1988). Furthermore, this compound has been reported as highly effective in both preventing and treating saprolegniasis in certain freshwater fish, including salmonid species (Pottinger and Day, 1999; Braidwood, 2000; Branson, 2002; Aller-Gancedo and Fregeneda-Grandes, 2007; Oono et al., 2007, 2008a, 2008b; Piamsomboon et al., 2013). Recently, Pyceze, containing the active ingredient bronopol, is the sole drug approved for aquaculture use in Japan, but it has experienced unstable supply. Given these limitations, the development of an alternative chemical solution is necessary to effectively prevent saprolegniasis in fish egg.

Glutaraldehyde (GA) is a dialdehyde compound utilized as a disinfectant and sterilant. GA possesses biocidal activity against all varieties of microorganisms including bacteria, mycobacteria, fungi, and viruses (Stonehill et al., 1963; Gorman et al., 1980; Russell, 1994). Due to its extensive biocidal activity, which results from its preference to cross-link with NH₂ groups on protein chains such as lysine amino acids in microbial cell walls, GA exhibits powerful antibacterial properties (McDonnell and Russell, 1999). In their research on saprolegniasis, Marking et al. (1994) showed the inhibitory effect of GA at a 50 mg/L concentration on S. parasitica and S. hypogyna growth compared to its chemical toxicity against salmonid fish. However, due to concerns regarding GA’s carcinogenic potential, the authors did not perform detailed condition studies. Currently, there is no evidence suggesting that GA possesses carcinogenic characteristics (Takigawa and Endo, 2006). GA has been documented to cause irritation to the skin and eyes, as well as skin sensitization and respiratory sensitization. However, there is no definitive evidence of reproductive toxicity, genetic toxicity, or neurotoxicity, and it is classified as “not classified” under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) in Japan (MHLW, 2015). GA, possessing these characteristics, has a long history of use as a disinfectant for medical devices and animals. Therefore, the potential use of GA to control saprolegniasis in aquaculture is considerable.

This study aims to assess the in vitro efficacy and mechanism of GA against the growth of Saprolegnia species. To assess the efficacy in detail, we exposed the Saprolegnia species to GA under two conditions: (i) general exposure, where the species were subjected to GA at various water temperatures and then cultured at their optimum temperature; and (ii) conditions mimicking aquaculture, where the species were exposed to GA and incubated at a constant temperature. We demonstrated GA’s efficacy on three Saprolegnia species encompassing seven strains, comparing it with malachite green, formaldehyde, and bronopol at various exposure times and culture temperatures. Furthermore, we scrutinized GA’s fungicidal effect relative to other aldehyde compounds, aiming to elucidate its potency against Saprolegnia species based on the aldehyde group. Finally, we present GA as a potential new alternative reagent to prevent saprolegniasis in aquaculture.

Materials and Methods

Strains of Saprolegnia species

Seven strains of Saprolegnia species were tested in this study: S. parasitica (NBRC8978, NBRC32708, NJM8604 (Hatai et al., 1990), NJM9868 (Hussein et al., 2001)), S. diclina (NBRC32710 (Hatai et al., 1990), NJM0246 (Miura et al., 2009)), and S. hypogyna (NBRC32711). These strains were cultured on sterile petri dishes (90 mm diameter) containing 20 mL of GY agar (Hatai and Egusa, 1979) at 15°C. Subsequently, the agar plugs containing hyphae (5.0 mm diameter) were transferred to petri dishes (90 mm diameter) with 20 mL of GY agar and incubated at 15°C for 48 h. The resulting colonies with a diameter of 5.0 mm were obtained as an agar plug using a cork borer and used for the subsequent chemical exposure.

Chemicals

Malachite green (FUJIFILM Wako Pure Chemical Corporation), formaldehyde solution (37% w/w, FUJIFILM Wako Pure Chemical Corporation), a commercial product containing 50% w/v bronopol (Pyceze; Elanco Japan Co., Ltd.), and a 25% w/w GA solution (FUJIFILM Wako Pure Chemical Corporation) were diluted to the following concentrations using distilled water: malachite green at 0.38, 0.75, 1.5, and 3.0 mg/L; formaldehyde at 81, 162, 324, and 648 mg/L; bronopol at 25, 50, 100, and 200 mg/L and GA at 27, 53, 106, and 212 mg/L.

Additionally, aldehyde compounds including 40% w/w of glyoxal (FUJIFILM Wako Pure Chemical Corporation), acetaldehyde (90% w/w, FUJIFILM Wako Pure Chemical Corporation), isobutyraldehyde (FUJIFILM Wako Pure Chemical Corporation), malonaldehyde bis (dimethyl acetal) (97% w/w, Merck KGaA), and valeraldehyde (97% w/w, Thermo Fisher Scientific Inc.) were prepared at various concentrations by serial two-fold dilution: glyoxal and acetaldehyde in distilled water, isobutyraldehyde in 2.5% dimethyl sulfoxide (DMSO) (FUJIFILM Wako Pure Chemical Corporation), and malonaldehyde bis (dimethyl acetal) and valeraldehyde in 10% ethanol (99.5% w/w, FUJIFILM Wako Pure Chemical Corporation).

The final concentrations ranged from 3,562 to 28,493 mg/L for glyoxal, 1,778 to 14,220 mg/L for acetaldehyde, 1,975 to 15,800 mg/L for isobutyraldehyde, 3,465 to 27,720 mg/L for malonaldehyde bis (dimethyl acetal), and 203 to 1,620 mg/L for valeraldehyde.

Experiment 1: The chemical effects on Saprolegnia growth under exposure to various temperatures and incubation at the strain’s optimum temperature

The following in vitro experiments were conducted according to Marking et al. (1994) with the following modification. Agar plugs containing individual hyphae from different strains of Saprolegnia listed above were exposed in triplicate to 30 mL of various reagents at different concentrations at 10, 15, and 20°C for 15, 30, and 60 min. Similarly, exposure of Saprolegnia strains to sterile distilled water was performed as a negative control. The agar plugs were rinsed three times with 30 mL of sterile distilled water and then placed on GY agar in the petri plates. The treated Saprolegnia strains were cultured at 15°C. At 7 days post-initiation of culture, the presence/absence of mycelium growth was observed. The minimum fungicidal concentration (MFC) was defined as the lowest dose at which no mycelial growth was observed among the triplicates.

Experiment 2: The chemical effects on Saprolegnia growth: Exposure and culture at varied temperatures mimicking treatment conditions in aquaculture

The exposure and culture of Saprolegnia species were carried out following the procedure outlined in Experiment 1, with the following modification: the Saprolegnia species were exposed to diluted chemicals and cultured at the same temperature (10 and 20°C).

Experiment 3: Efficacy of various aldehyde compounds on growth of Saprolegnia parasitica NJM 8604

S. parasitica NBRC 8604 was exposed to different concentrations of glyoxal, acetaldehyde, isobutyraldehyde, malonaldehyde bis (dimethyl acetal) and valeraldehyde. The exposure and culture procedures were conducted as described in experiment 1, with the exception of the exposure temperature (15°C). Additionally, exposure of S. parasitica NBRC 8604 to the corresponding solvent was performed as a negative control.

Results

Experiment 1: The chemical effects on Saprolegnia growth under exposure to various temperatures and incubated at the strain’s optimum temperature

The MFC values of chemicals for the tested Saprolegnia strains, exposed to various temperatures (10, 15, and 20°C) and then incubated at 15°C, are shown in Table 1. The MFC ranged from 0.75 to 3.0 mg/L for malachite green and 81 to 648 mg/L for formaldehyde across all tested groups. In contrast, the bronopol- and GA-treated groups exhibited a MFC range of 50 to 200 and 53–212 mg/L, respectively, with the exception of two groups; treatment with bronopol and GA at 10°C for 15 min against S. parasitica NBRC32708 did not inhibit its growth. When comparing the MFC among groups where the same Saprolegnia strains were exposed to identical reagents at the same temperature, it was observed that the MFCs either decreased or remained unchanged as the exposure time increased. No tendency towards higher MFCs for specific Saprolegnia strains was observed among the tested reagents. Mycelium growth was observed in all groups exposed to distilled water (negative control groups).

Table 1. The minimum fungicidal concentration (MFC) of tested chemicals for seven Saprolegnia strains exposed at 10, 15, and 20°C and incubated at 15°C

Chemicals	Exposure temperature (°C)	Exposure time (min)	MFC (mg/L)*
			S. parasitica				S. diclina		S. hypogyna
			NBRC8978	NBRC32708	NJM9868	NJM8604	NBRC32710	NJM0246	NBRC32711
Glutaraldehyde	10	15	212	>212	212	106	212	212	106
		30	106	212	212	106	212	106	53
		60	53	106	106	53	53	106	53
	15	15	53	106	106	212	212	106	212
		30	53	106	106	212	53	106	106
		60	53	53	106	212	53	106	106
	20	15	106	212	106	212	212	212	106
		30	53	106	106	106	106	106	106
		60	53	106	106	106	106	106	53
Malachite green	10	15	1.5	3.0	1.5	3.0	3.0	3.0	3.0
		30	0.75	1.5	1.5	1.5	1.5	1.5	1.5
		60	0.75	1.5	1.5	0.75	1.5	1.5	0.75
	15	15	1.5	3.0	1.5	1.5	1.5	1.5	1.5
		30	1.5	3.0	1.5	0.75	1.5	1.5	1.5
		60	0.75	1.5	0.75	0.75	0.75	1.5	0.75
	20	15	1.5	3.0	0.75	1.5	1.5	1.5	1.5
		30	1.5	1.5	0.75	1.5	0.75	0.75	0.75
		60	0.75	0.75	0.75	0.75	0.75	0.75	0.75
Fomaldehyde	10	15	324	324	324	648	324	324	324
		30	162	324	324	324	324	324	324
		60	162	324	324	162	324	162	162
	15	15	324	324	324	162	324	324	324
		30	162	324	324	81	324	324	324
		60	162	162	162	81	162	162	324
	20	15	162	324	324	324	162	324	324
		30	162	162	162	162	81	162	162
		60	81	162	162	162	81	162	162
Bronopol	10	15	200	>200	200	200	200	200	200
		30	200	200	200	200	200	100	100
		60	100	100	100	100	100	100	100
	15	15	100	200	50	200	200	100	100
		30	50	200	50	200	200	100	50
		60	50	100	50	100	100	50	50
	20	15	100	100	200	100	100	100	100
		30	50	50	100	100	100	50	50
		60	50	50	100	100	50	50	50

*  MFC was calculated by exposure of triplicate agar plug with tested strains.

Experiment 2: The chemical effects on Saprolegnia growth: Exposure and culture at varied temperatures mimicking treatment conditions in aquaculture

The MFC values of chemicals for the tested Saprolegnia strains, exposed and cultured at various temperatures (10 and 20°C) to mimic the treatment conditions in aquaculture, are shown in Table 2. Malachite green exhibited an MFC of less than 3.0 mg/L for all tested Saprolegnia strains in all treatment groups, regardless of exposure time and strain variation. Formaldehyde treatments showed MFCs ranging from 81–324 mg/L. Notably, exposure to bronopol at 10°C and 20°C for 15 min did not inhibit the growth of S. diclina NJM0246. Similarly, the treatment at 20°C was ineffective against S. parasitica NBRC32708, while other bronopol-treated groups exhibited MFCs ranging from 25–200 mg/L. In the case of GA treatment, most groups showed MFCs ranging from 53–212 mg/L, except for one group exposed to S. parasitica NJM9868 at 20°C for 15 min.

Table 2. The minimum fungicidal concentration (MFC) for seven Saprolegnia strains exposed at 10 and 20°C and incubated at 10 and 20°C, respectively, to mimic the treatment conditions in aquaculture farms.

Chemicals	Exposure and incubation temperature (°C)	Exposure time (min)	MFC (mg/L)*
			S. parasitica				S. diclina		S. hypogyna
			NBRC8978	NBRC32708	NJM9868	NJM8604	NBRC32710	NJM0246	NBRC32711
Glutaraldehyde	10	15	106	212	212	212	106	212	212
		30	106	106	106	106	106	106	212
		60	106	53	53	53	106	106	106
	20	15	212	212	>212	212	106	212	212
		30	106	106	106	212	106	106	212
		60	106	106	106	106	106	106	106
Malachite green	10	15	1.5	3.0	3.0	1.5	1.5	1.5	3.0
		30	0.75	3.0	1.5	1.5	0.75	1.5	1.5
		60	0.75	3.0	1.5	1.5	0.75	1.5	1.5
	20	15	1.5	1.5	1.5	1.5	1.5	1.5	1.5
		30	0.75	0.75	1.5	0.75	0.75	0.75	0.75
		60	0.75	0.75	1.5	0.75	0.75	0.75	0.75
Fomaldehyde	10	15	324	324	324	324	324	324	324
		30	162	324	162	324	162	162	162
		60	162	162	162	162	162	162	162
	20	15	162	324	324	162	162	162	324
		30	162	162	162	81	162	81	324
		60	81	162	162	81	81	81	162
Bronopol	10	15	200	200	100	200	200	>200	100
		30	50	200	100	100	200	200	100
		60	25	200	100	100	200	200	50
	20	15	200	>200	200	200	100	>200	100
		30	50	200	200	200	100	100	100
		60	50	200	100	200	100	100	100

*  MFC was calculated by exposure of triplicate agar plug with tested strains.

A time-dependent reduction in MFC was observed across groups with the same agents, Saprolegnia strains, and temperatures. Exposure to distilled water did not inhibit the mycelium growth in any of the treatment groups, serving as the negative control groups.

Experiment 3: Efficacy of various aldehyde compounds on growth of Saprolegnia parasitica NJM 8604

The strains of interest were exposed to various aldehyde compounds using the same method as in Experiment 1. All tested reagents inhibited the growth of S. parasitica NJM8604 across all exposure groups (Table 3). The MFC for all groups ranged from 405 mg/L to 28,493 mg/L. Valeraldehyde exhibited the lowest MFC at each exposure time (810 mg/L in 15 min and 405 mg/L in 30 and 60 min) among the tested reagents. MFC either decreased or remained unchanged as the exposure time increased when comparing groups of the same agents at 15°C. Exposure to the solvents (distilled water, 10% ethanol, and 2.5% DMSO) resulted in mycelium growth in all treatment groups (negative control groups).

Table 3. The minimum fungicidal concentration (MFC) of aldehyde compounds for Saprolegnia parasitica NJM 8604 exposed and incubated at 15°C

Chemicals	Exposure time (min)	MFC (mg/L)*
Glyoxal	15	28,493
	30	28,493
	60	14,246
Acetaldehyde	15	14,220
	30	7,110
	60	7,110
Isobutaldehyde	15	15,800
	30	7,900
	60	7,900
Malonaldehyde bis (dimethyl acetal)	15	27,720
	30	27,720
	60	13,860
Valeraldehyde	15	810
	30	405
	60	405

*  MFC was calculated by exposure of triplicate agar plug with tested strains.

Discussion

Saprolegniasis is frequently observed in aquaculture. The causative pathogen encompasses several species of the Saprolegnia genus, specifically including S. parasitica, S. diclina, and S. hypogyna (van West, 2006; Fregeneda-Grandes et al., 2007; van Den Berg et al., 2013; Liu et al., 2017). In the present study, we used these species, including seven strains that could be prepared. El Gamal et al. (2023) observed occurrences of Saprolegnia outbreaks in Nile tilapia farms during the winter season, when the water temperatures ranged from 15.5 to 18.5°C. Saprolegnia growth is influenced by the temperature of rearing water: Kitancharoen et al. (1996) suggested that low temperatures, such as 10°C, promote mycelial growth and zoospore development in Saprolegnia diclina and S. parasitica strains. Cold-water fish, including salmonid species, are commonly cultivated within the temperature range of 10–20°C in aquaculture practices (Brett et al., 1969; Koskela et al., 1997; Ineno et al., 2005; Hvas et al., 2017; Bockus et al., 2021; Mishra et al., 2021; Steiner et al., 2022; Lee and Balasubramanian, 2023). Consequently, the temperature range for the exposure and incubation of Saprolegnia species in the present study was determined to be between 10°C and 20°C. So far, many studies have evaluated the effect of reagents on growth of Saprolegnia by culturing them at optimal temperatures for Saprolegnia species after exposure to various temperatures (Marking et al., 1994; Oono and Hatai, 2007; Tedesco et al., 2019). Meanwhile, in aquaculture, eggs are typically treated by disinfectants dissolved in rearing water without adjusting the temperature, as it can be challenging to maintain or obtain water at multiple temperature levels at aquaculture sites. The water temperature at which Saprolegnia grows (i.e. the temperature at which the fish are reared) is not always optimal. Therefore, in our study, we employed both approaches. In Experiment 1, we set the culture temperature of Saprolegnia to its optimum level. However, the experimental approach replicating aquaculture conditions for Saprolegnia exposure is quite limited. Thus, in Experiment 2, we matched the treatment and culture temperatures to mimic the treatment conditions in aquaculture farms.

In both experiments (Experiment 1 and 2), the MFC of malachite green and formaldehyde for the tested species ranged from 0.75–3.0 mg/L and 81–648 mg/L, respectively. Malachite green was previously used at 5 mg/L for 60 min at 12°C to prevent the outbreaks of saprolegniasis (Marking et al., 1994). Additionally, in hatcheries, formaldehyde is utilized at concentrations ranging from 250 to 1,667 mg/L at 11–30°C for disinfection of fish eggs (Marking et al., 1994; Walser and Phelps, 1994; Barnes et al., 2000, Barnes and Soupir 2007). Consequently, MFC for malachite green and formaldehyde calculated in the current study further support the effectiveness of the previously available treatment, even though their use is currently not permitted in the aquaculture industry in many countries.

Sales company Elanco, which produces Pyceze, a commercial product containing bronopol as an active ingredient, recommends bathing salmon eggs daily in 50 mg/L of bronopol for 30 min to control saprolegniasis (https://elanco.co.jp/製品情報/魚/パイセス). In the case of rainbow trout and ayu eggs, Oono et al. (2007, 2008a) effectively managed saprolegniasis occurrence by exposing the eggs to 50 and 100 mg/L of bronopol daily for 30 min. However, a single of 30-min exposure to bronopol in our present study resulted in an MFC exceeding 100 mg/L under all experimental conditions. Oono and Hatai (2007) also observed that the MIC of bronopol against S. parasitica, S. diclina, and other Saprolegnia species exceeded Elanco’s recommended condition (50–100 mg/L) at a temperature of 20°C during a 30-min exposure. Therefore, repeated bronopol treatments (approved condition in Japan: i) 50 mg/L for 30 min daily, or ii) 100 mg/L for 30 min every other day or every third day; MAFF, 2024) may be necessary to prevent saprolegniasis in fish eggs. In the present study, temperature did not significantly influence bronopol’s efficacy against Saprolegnia growth; however, it exhibited effective anti-saprolegniasis properties across a wide temperature range. Additional comprehensive studies encompassing a broader spectrum of temperatures and environmental conditions are necessary to obtain more conclusive evidence.

Exposing tested Saprolegnia species for more than 30 min completely inhibited their growth in both Experiment 1 and 2 under all tested conditions. Marking et al. (1994) also reported the efficacy of GA against only one strain each of S. parasitica and S. hypogyna in vitro, using a concentration of 50 mg/L for 60 min of exposure, supporting our assertion of GA as disinfectants for fish eggs. Moreover, the present study also indicated that GA can possibly control another Saprolegnia species, S. diclina. This suggests that GA has significant potential as an effective disinfectant against Saprolegnia species in both warm and cold-water fish aquaculture settings. Meanwhile, the time-dependent decrease in MFC by GA treatment, with the exception of one strain (NBRC8978), was not observed in Experiment 1 but was observed in Experiment 2 (e.g., exposure at 10°C). This result indicates that incubation temperature may influence the MFC value of S. parasitica. Further investigation should verify the effectiveness of GA for controlling saprolegniosis in actual aquaculture settings to account for environmental conditions and other influencing factors.

Thus far, the toxicity of GA against fish eggs has only been demonstrated in rainbow trout. Marking et al. (1994) conducted a study where rainbow trout eggs were exposed to GA for 60 min every other day at 12°C, from fertilization until hatching or for 2 weeks. The results indicated a significant toxic effect at a concentration of 300 mg/L GA or higher, whereas no significant toxicity was observed at concentrations ranging from 50 to 200 mg/L, as the hatching rates did not show a reduction of 10% or more compared to the control groups. This confirms that the maximum MFC value of GA in our study, at a concentration of 212 mg/L, may not be toxic to salmonid eggs. Further investigation is needed to evaluate the toxicity of GA against other fish species, particularly during developmental phases ranging from fertilization to the eyed stage, which are prone to water mold outbreaks.

Both GA and formaldehyde contain the aldehyde functional group in their molecular structure. To investigate whether their disinfection effect on Saprolegnia species stems from the aldehyde groups, this study examined five chemical compounds, each incorporating aldehyde groups. While all compounds tested showed fungicidal effects against S. parasitica (NJM 8604), it is noteworthy that their MFCs were significantly higher compared to those observed for formaldehyde and GA in Experiment 1. Among the aldehydes assessed in this study, acetaldehyde has been showed to inhibit the yeast-to-hypha conversion of Candida albicans, a pathogenic yeast in humans (Chauhan et al., 2011). Additionally, malonaldehyde has demonstrated significant antibacterial activity against Staphylococcus xylosus and Lactiplantibacillus plantarum (Zhang et al., 2022). However, despite these properties, their fungicidal efficacy against Saprolegnia species appears to be lower than that of formaldehyde and GA. GA and formaldehyde possess a smaller side chain attached to the aldehyde group compared to the other tested aldehyde compounds (GA: https://pubchem.ncbi.nlm.nih.gov/compound/3485, formaldehyde: https://pubchem.ncbi.nlm.nih.gov/compound/712, glyoxal: https://pubchem.ncbi.nlm.nih.gov/compound/7860, acetaldehyde: https://pubchem.ncbi.nlm.nih.gov/compound/177, isobutyraldehyde: https://pubchem.ncbi.nlm.nih.gov/compound/6561, malonaldehyde bis (1,1,3,3-tetramethoxypropane): https://pubchem.ncbi.nlm.nih.gov/compound/66019, valeraldehyde: https://pubchem.ncbi.nlm.nih.gov/compound/8063). This smaller side chain may contribute to their strong fungicidal efficacy against S. parasitica.

In conclusion, the present study demonstrated that GA successfully inhibits the growth of Saprolegnia at concentrations ranging from 53 to 212 mg/L, with a minimum exposure duration of 30 min. The treatment time-dependent change of sensitivity to GA is likely influenced by incubation temperature. These findings suggest its potential as a prospective alternative treatment to the traditionally used chemicals such as malachite green, formaldehyde, and bronopol to prevent saprolegniasis. A comparison with the efficacy of aldehyde derivatives revealed the possibility that the small side chains bound to the aldehyde group are related to the fungicidal effect of GA on Saprolegnia species.

Acknowledgment

We are grateful to Dr O. Kurata from Nippon Veterinary and Life Science University, Tokyo, Japan for sharing the Saprolegnia strains. This work was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan and JSPS KAKENHI Grant number 23K05381.

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