Fungal infections (mainly Saprolegnia sp.) are prevalent in hatcheries and may affect hatching of fish eggs. Several chemical agents available to control these infections have remained low in efficacy. Several laboratory studies indicate that nanoparticles are used to control of several strains of fungi. This study evaluated efficacy of sodium chloride (NaCl), hydrogen peroxide (H2O2), zinc oxide nanoparticle (ZnONP) and copper nanoparticle (CuNP) against Saprolegnia spp. in Clarias gariepinus eggs. Although all the test agents were effective in inhibiting Saprolegnia spp. growth by at least 50% at concentration ranges ≥ 1000 ppm, significantly (P < 0.05) higher Saprolegnia sp. spore reduction was achieved using CuNP and ZnONP compared to NaCl and H2O2 at test concentration between 500 and 1000 ppm. Also significantly (P < 0.05) higher hatchability was achieved using CuNP and ZnONP compared with NaCl and H2O2 at concentration ranges of 500 and 1000 ppm. This study demonstrates that antifungal properties of ZnONP and CuNP render them good alternative or addition to the commonly used antifungal agents such as common salt and hydrogen peroxide. Based upon further safety evaluation, these nanoparticles should be considered in control of fungal infections of fish eggs.

Ngugi CC, Oyoo-Okoth E, Odhiambo CO (2021) Efficacy of sodium chloride, hydrogen peroxide, Cu and ZnO nanoparticles on hatchability and control of Saprolegnia spp. on Clarias gariepinus eggs. Ann Aquac Res 6(1): 1049.

•    Nanoparticles
•    Zinc oxide NP
•    CuNP
•    Saprolegnia
•    Fish Disease

The world’s demand for fish is steadily i resulting in increased production of more farmed fish per unit of land and water. (1,2,3,4) Hatcheries managers have responded to the challenge by supplying an ever-increasing amount of fingerlings (5, 6). Increased egg loading densities are now an obviously features in fish hatcheries, leading to increased exposure to pathogens (7,8,9,10). During artifical propagation of eggs, fungal infections on eggs have been widely reported in hatcharies (11, 12, 13) .

Among the fungi, water molds (Class Oomycetes) of the genus Saprolegnia (order: Saprolegniales) remains one of the most ubiquitous fungal parasitic groups affecting fish eggs during artificial propagation (14, 15, 16, 17). Saprolegnia infection of fish eggs is widespread and has been reported in Nile tilapia Oreochromis niloticus (18) , Atlantic salmon, Salmo salar L (17, 19), common carp, Cyprinus carpio (20) , rainbow trout Oncorhynchus mykiss (15, 16, 21), silver crucian carp, Carassius carassius (22), African catfish Clarias gariepinus (23) and Indian Carps Labeo rohita (25) among others. Infections begin with the settling of zoospores on dead eggs during incubation, then adherence to the membrane of fish eggs, and eventual forceful penetrate into the egg, spreading the infection to the eggs. If left untreated, they spread over the entire egg mass and cause egg mortalities by hyphal breaching of the chorionic membrane (17, 26). Therefore efforts aimed at controlling the pathogens during egg incubation remains a priority (8) since outbreaks may result in the loss of entire batches of eggs (27) or reductions in egg hatchability.

Healthy embryos and appropriate incubation conditions, including tedious removal of dead fish eggs and suitable water chemistry, help in the reduction of the disease (28). Routine application of disinfectants is common during egg incubation in fish hatcheries worldwide (29). In the past malachite green and formalin were the most potent fish fungicide used in a fish hatchery (30) . Malachite green was later established to be toxic and a potential carcinogen, teratogen and mutagen; hence, its usage was banned in fish intended for human consumption (31, 32). Formalin as a disinfectant, has also been banned in most countries (33). Therefore search for acceptable safe/efficient alternatives have progressed including the less effective iodine (34, 35) Investigations to date have been carried out principally on hydrogen peroxide (H2 O2 ), sodium chloride (NaCl) and ozone (O3 ). Hydrogen peroxide, at concentrations of 500–1000 ppm, has been documented to be effective against Saprolegnia spp. infections in salmonid and African catifish eggs (36, 37, 38) Nevertheless, increasing concentrations of hydrogen peroxide in the water was reported to decrease the hatch rate of O. mykiss eggs (39). In the case of sodium chloride, there seems to be a general agreement that it is not as effective as hydrogen peroxide in controlling infections (40). Alternative strategies have looked at the use of ozone (O3 ) (41) as a disinfectant in trout hatcheries where it was found to control Saprolegnia spp. outbreaks but significantly reduced the hatching rates of fish eggs (42).

Novel nanatochnology is increasingly applied in different sciences to control fungal infection, despite the slow progress in adoption and application in aquaculture (43, 44, 45). Nano-sized particles have properties that differ from the larger particles of the same substance, which are consequences of cutting the size of particles so as to increase their activity (46). Currently the use of nanosilver, zinc oxide and copper nanoparticles have applications in various industries such as agriculture, livestock, household, military and human medicine due to their antimicrobial properties (47). They have been found to be effective in removing a wide range of fungi in several plant and animals tissues (48). However, their applications in disease control in aquaculture remains low. To date, copper nanoparticle has been applied to control the growth of fungi isolated from Rutilus frisii kutum eggs (49) while nanosilver has been used to enhance hatchability of O. mykiss egg (50). However, efficiency of these nanoparticles relative to the conventionally and widely used antifungal treatments have not been investigated. Therefore this study evaluated the antifungal efficacy of ZnO and Cu nanoparticles against conventional treatment agents (NaCl and H2 O2 ) in controlling Saprolegnia spp. and hatchability Clarias gariepinus eggs.

Experimental facility and fertilized eggs

The entire experiment was carried out in a hatchery at Mwea Fish Farm (Latitude 0?36.73’S, and longitude 37?22.84’E). Ultra violet treated water obtained from a well was used for the current experiment. Temperature and dissolved oxygen (DO) were measured using probes of the oxygen–temperature meter (Model 55, YSI, Yellow Springs Ohio, USA), while pH was determined using a pH meter (Hanna Instruments, Model 8519, USA). Total ammonia nitrogen (TAN) measured using the method of (Boyd and Tucker, 1992). The mean temperature was 26.5 ± 1.1°C, total hardness as CaCO3 70.6 ± 6.7 mg/L, pH 6.7 ± 0.4, DO 6.1 ± 0.5 TAN 0.28 ± 0.04 mg/L. The water flow rate was maintained at 1.01 ± 0.02 liter/min with aeration provided throughout the incubation and hatching period using an electric pump.

Eggs used in this study were obtained from artificial reproduction of C. gariepinus detailed in (51). To induce spawning, a female weighing 360 g was selected and injected with pituitary suspension from a sacrificed ripe male weighing 354 g. After 12 h, the eggs were stripped into a dry bowl and fertilized with milt from a ripe male.

Test materials

To allow for comparative evaluation of different efficacies, the same test concentrations of 4000, 2000, 1000, 500, 250, 100 against control (0 ppm) were used during the study. The concentrations were made through dilution of the test agents. Copper nanoparticles (CuNP) (Nanostructured Avizheh Company) and ZnONP suspensions with NP size of 70 ± 15 nm (Ward Hill, MA, USA) were the nanoparticles used in this study. The CuNP and ZnONP suspension were then diluted with potato dextrose agar (PDA, containing the extract from 200 g boiled potato, 20 g glucose and 20 g agar in 1 l of distilled water). Nacl and H2 O2 were diluted in distilled water.

Fungal isolation and culture

Source of Saprolegnia sp. used to evaluate antifungal activity of copper nanoparticles was isolated from eggs of Clarias gariepinus at the Department of Microbiology, University of Eldoret. They were propagated on 40 g yeast extract glucose chloramphenicol agar (YGC) powder and dissolved in 1 liter of distilled water. Fungal colonies were placed on slides containing Lactophenol cotton blue (LPCB), covered with a cover slip and identified using morphological characterization by observation of their sporangia under a microscope. The fungal isolates were identified using available identification keys. Agar plates were stored at 4°C until used.

Antifungal test

Antifungal tests were performed by the agar dilution method. The test agents at different concentration were poured into the Petri dishes (9 cm diameter). 1 mL of Saprolegnia sp. culture solution estimated to contain approximately 8 × 105 zoospores mL-1 were taken from the fungal cultures and placed in the center of each Petri dish. The number of Saprolegnia sp. spores was determined using a haemocytometer. The Petri dishes with the inoculums were then incubated at 25°C.

The mycelia growth index was determined using the formula:

Hatchability experiments

After fertilization, the eggs were randomly counted into equal lots of 50-eggs and each lot spread on 5 cm × 5 cm strips of mosquito netting where the eggs attached due to the natural adhesiveness of catfish eggs. Each strip containing eggs was then inserted inside individual 15 cm × 15 cm hatching bags made from fine meshed mosquito netting (mesh size 0.5 mm) which would prevent escape of any hatched larvae. The individual hatching bags were randomly assigned in triplicate to static bath treatments of given concentrations of the four test agents for 60 minute exposure periods before being transferred to randomized compartments of the incubation tank. After 24 h the hatching bags were removed from the incubation tank, and evaluated for the number of dead eggs using a dissecting microscope (Olympus SZ40, Olympus, London, UK) at ×4 magnification. Hatchability (% hatch) was calculated by dividing the number of larvae by the total number of eggs per lot and multiplying by 100 (i.e. larvae/50?100).

Data analysis

In this study, all the response variables are binary in nature and naturally follow binomial distribution. Therefore the relationship between the response variables and factors were modeled using Probit analysis, which is a form of regression using GraphPad Prism 6.0 Statistical Software. Maximum likelihood was used to estimate the regression coefficient (R square). During Probit analysis, all data were transformed to Log Base 10 to linearize the relationship between the response variables and factors. For each analysis, the response frequency was observed as response variables from a total observation of 50 eggs. Meanwhile the concentrations of the test agents was the covariate. The resulting probability outcomes were multiplied by 100 to determine the expected percent of the response frequency. To test for the significance of the Probit plots, Z statistics was calculated. Significant differences were verified using P-value at 0.05. The modeled fit was confirmed using chi-square goodness of fit test between the observed response values and predicted probability of response values. The resultant graphs plotted consisted of Probits of response variables (spore counts and hatchability) in Y-axis and test chemical concentration in X-axis.

This study did not require any ethical approval.

Antifungal effect of test agents

In this trial, the spore numbers for all the test agents over the experimental period are shown in (Figure 1) while the calculated spore reduction index (%) is shown in (Figure 2). Based on the regression plots, all the test agents were effective in inhibiting Saprolegnia sp. fungal growth by at least 50% at concentration ranges ≥ 1000 ppm. The Saprolegnia spp. spore reduction at various concentrations of the test agents followed a dose response with good model fit (P < 0.05, (Table 1)). After 24 hours of exposure, maximum percentage of Saprolegnia sp. spore reduction (93%) was achieved using CuNP at test concentration 500 and 1000 ppm. Meanwhile up to 81% pore reduction occurred in ZnONP exposure at exposure doses of 1000 to 1500 ppm. Exposure to NaCl and H2 O2 resulted in a maximum spore reduction of 50 to 65% at test concentration of 1000 to 2000 ppm.

The estimated hatchability of the C. gariepinus eggs after exposure to different concentrations of test agents are shown in Fig. 3. Based on the exposure concentrations tested, the hatchabilities of all the test agents followed a dose response with good model fit (P < 0.05, (Table 2)). A maximum hatchability of 86 to 98% occurred in CuNP (at concentration ranges of 500 to 2000 ppm), which was followed by hatchability of eggs in ZnONP (84 to 92%) at concentration ranging between 500 to 2000 ppm. Treatment using NaCl at concentration ranges between 1000 to 2000 ppm resulted in maximum hatchability of ranges between 65% to 75%, while similar concentration ranges of H2 O2 resulted in maximum hatchability of 67% to 72%.

Fungus belonging to Saprolegnia order or other orders may cause serious losses in fish hatcheries (52). Therefore finding suitable antifungal agents would help reduce losses in hatcheries during fingerling production. The major aim of this study was to investigate the antifungal efficacy of ZnO and Cu nanoparticles against conventional treatment agents (NaCl and H2 O2 ) in controlling Saprolegnia spp. and hatchability Clarias gariepinus eggs. Generally, we established that all the test agents effectively inhibited Saprolegnia sp. fungal growth by at least 50% at concentration ranges of ≥ 800 ppm. Several studies have indicated that NaCl and H2 O2 are effective antifungal agents but their efficacies are somewhat low. In several studies improved hatch due to salt treatment typically range from 500–4000 ppm as NaCl tend to be toxic beyond 5000 ppm (53, 54). In this study we achieved up to 93% spore reduction after 24 hours of exposure in CuNP at test concentration 500 and 1000 ppm. This agrees with observation of (49). Nevertheless, antifungal activity of copper nanoparticles against selected pathogenic fungi has been reported in plants (55, 56). Meanwhile upto 81% spore reduction occurred at ZnONP exposure doses of 900 to 1500 ppm. The current efficacious ranges are higher when compared with study of the inhibition of antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum estimated at 245 ppm (57). Subsequently that study recommended ZnONP as an effective fungicide in agricultural and food safety applications. In another study, the minimum inhibitory concentration of ZnO nanoparticle against Saccharomyces cerevisiae, Candida albicans, Aspergillus niger, and Rhizopus stolonifer was about 100 ppm (58). It is probable that the Zno and Cu nanoparticles causes changes in the structure and function of the fungi cell as well as disrupt the replication and transcription DNA leading to death of fungal microorganisms . ZnO and CuNPs display show great enhancement in the antimicrobial activity due to their unique properties such as large surface area.

Control of fungal growth is important in aquaculture if the resultant hatchability of the eggs can improve. In this study were tested the hatchability of C. gariepinus eggs following treatments using the test agents. Our estimated maximum hatchability of 86 to 98% occurred in CuNP (at concentration ranges of 500 to 2000 ppm), which was followed by hatchability of eggs in ZnONP (84 to 92%) at concentration ranging between 500 to 2000 ppm. Although there are few studies available on the use of nanoparticles in enhancing hatching rates, there are a number of studies that have indicated that once fungal infections on the eggs are reduced, then hatching probability can be improved. Therefore the lower eggs hatchability using NaCl and H2 O2 at concentration ranges between 1000 to 2000 ppm could be associated with inability of these test agents to totally eliminate pathogens on the eggs. Based on the results obtained from this study, it is clear that that copper and zinc oxide nanoparticles prevent the growth of the fungus Saprolegnia sp. in vitro. Consequently, the zinc oxide and copper nanoparticles can be used as a treatment to prevent the growth of fungus Saprolegnia sp.

In conclusion, ZnO and Cu nanoparticles have antifungal effects on a Saprolegnia sp. isolated from C. gariepinus fish eggs and enhanced hatchability of the fish eggs. Antifungal effects are dependent on concentration used. Further research is required to find the most appropriate way of using these substances in aquaculture activities. Further research is required on the exact mechanism of action of copper and zinc oxide nanoparticles on aquatic pathogens and egg biology. Therefore the next steps would be to test what concentration of copper and zinc nanoparticles fish can tolerate together with safety in their use. However, for resource people, the use of other conventional antifungal agents such as common salt is not discouraged but should be used with knowledge that they are not as effective as the copper and zinc oxide nanoparticles.

Table 1: Statistical test of significance for the spore reduction during the study.

Table 2: Statistical test of significance for hatchability of C. gariepinus eggs following exposure to different concentration of antifungal agents during the study.

We would like to thank Mwea Fish Farm for allowing their facilities and hatchery to conduct this study. We also wish to express our gratitude to Department of Microbiology, University of Eldoret for fungal isolation and culture and permission to use their facilities and staff.

Data will be available on request from the corresponding author.

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