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Annals of Aquaculture and Research

Efficacy of sodium chloride, hydrogen peroxide, Cu and ZnO nanoparticles on hatchability and control of Saprolegnia spp. on Clarias gariepinus eggs

Original Research | Open Access

  • 1. School of Natural Resources and Environmental Studies, Department of Natural Resources, Karatina University
  • 2. School of Environmental Studies, Department of Environmental Biology, University of Eldoret, School of Natural Resources and Environmental Studies, Karatina University
  • 3. Department of Zoological Sciences, Kenyatta University
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Corresponding Authors
Elijah Oyoo-Okoth, School of Environmental Studies, Department of Environmental Biology, University of Eldoret, School of Natural Resources and Environmental Studies, Karatina University, 1125, Eldoret, Kenya Tel No: +254720222082
Abstract

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.

Citation

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.

Keywords

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

INTRODUCTION

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.

MATERIALS AND METHODS

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.

ETHICS STATEMENT

This study did not require any ethical approval.

RESULTS

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%.

DISCUSSION

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.

    Probit parameter estimates Chi-square goodness of fit test
Test agent R square Z P-value χ2 P-value
CuNP 0.9567 13.8104 0.0000 46.0452 0.0001
ZnONP 0.9634 8.1735 0.0032 21.3411 0.0043
NaCl 0.9876 10.1892 0.0002 54.4322 0.0001
H2 O2 0.8734 10.6501 0.0001 43.7517 0.0012

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

    Probit parameter estimates Chi-square goodness of fit test
Test agent R square Z P-value χ2 P-value
CuNP 0.9911 23.9995 0.0000 43.12323 0.0000
ZnONP 0.9773 12.1094 0.0003 9.4411 0.0002
NaCl 0.9811 16.0023 0.0000 11.4339 0.0000
H2 O2 0.9477 15.34463 0.0000 23.7520 0.0000

 

ACKNOWLEDGEMENTS

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 AVAILABILITY STATEMENT

Data will be available on request from the corresponding author.

REFERENCES

1. Béné C, Arthur R, Norbury H, Allison EH, Beveridge M, et al. Contribution of fisheries and aquaculture to food security and poverty reduction: assessing the current evidence. World Development. 2016; 79: 177-196.

2. Little DC, Newton R, Beveridge M. Aquaculture: a rapidly growing and significant source of sustainable food? Status, transitions and potential. Proc Nutr Soc. 2016; 75: 274-286.

3. Konikoff M. Introduction to the general principles of aquaculture. Routledge. 2017.

4. Lima LB, Oliveira FJM, Giacomini HC, Lima?Junior DP. Expansion of aquaculture parks and the increasing risk of non?native species invasions in Brazil. Reviews in Aquaculture. 2018; 10: 111-122.

5. Gullian-Klanian M & Arámburu-Adame C. Performance of Nile tilapia Oreochromis niloticus fingerlings in a hyper-intensive recirculating aquaculture system with low water exchange. Lat. Am. J. Aquat. Res. 2017; 41: 150-162.

6. Nyonje B, Opiyo M, Orina P, Abwao J, Wainaina M, et al. Current status of freshwater fish hatcheries, broodstock management and fingerling production in the Kenya aquaculture sector. Livest Res Rural Dev. 2018; 30.

7. Lehtonen TK & Kvarnemo C. Density effects on fish egg survival and infections depend on salinity. Marine Ecology Progress Series. 2015; 540: 183-191.

8. De Swaef E, Van den Broeck W, Dierckens K, Decostere, A. Disinfection of teleost eggs: a review. Reviews in Aquaculture. 2016; 8: 321-341.

9. Pandit NP, Wagle R, Ranjan R. Alternative artificial incubation system for intensive fry production of Nile tilapia (Oreochromis niloticus). International Journal of Fisheries and Aquatic Studies. 2017; 5: 425- 429.

10. Rahman MA, Rahman MH, Yeasmin S, Asif A, Mridha D. Identification of causative agent for fungal infection and effect of disinfectants on hatching and survival rate of Bata (Labeo bata) larvae. Adv. Plants Agric. Res. 2017; 7: 00264.

11. Fregeneda?Grandes J, Rodríguez?Cadenas F, Aller?Gancedo J. Fungi isolated from cultured eggs, alevins and broodfish of brown trout in a hatchery affected by saprolegniosis. Journal of Fish Biology. 2007; 71: 510-518.

12. Bricknell I. Types of Pathogens in Fish, Waterborne Diseases, Fish Diseases. Elsevier. 2017; 53-80.

13. Novakov N, Mandi? V, Kartalovi? B, Vidovi? B, Stojanac N. Comparison of the efficacy of hydrogen peroxide and salt for control of fungal infections on Brown Trout (Salmo trutta) eggs. Acta Scientiae Veterinariae. 2018; 46: 5.

14. Ghiasi M, Khosravi A, Soltani M, Binaii M, Shokri H, et al. Characterization of Saprolegnia isolates from Persian sturgeon (Acipencer persicus) eggs based on physiological and molecular data. Journal de Mycologie Médicale/Journal of Medical Mycology. 2010; 20: 1-7.

15. Shahbazian N, Ebrahimzadeh Mousavi H, Soltani M, Khosravi A, Mirzargar S, et al. Fungal contamination in rainbow trout eggs in Kermanshah province propagations with emphasis on Saprolegniaceae. Iranian Journal of Fisheries Sciences. 2010; 9: 151- 160.

16. Fadaeifard F, Bahrami H, Rahimi E, Najafipoor A. Freshwater fungi isolated from eggs and broodstocks with an emphasis on Saprolegnia in rainbow trout farms in west Iran. African Journal of Microbiology Research. 2011; 5: 3647-3651.

17. Thoen E, Evensen Ø, Skaar I. Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs. Journal of Fish Diseases. 2011; 34: 601-608.

18. Borisutpeth P, Kanbutra P, Hanjavanit C, Chukanhom K, Funaki D. Effects of Thai herbs on the control of fungal infection in tilapia eggs and the toxicity to the eggs. Aquaculture Science. 2009; 57: 475-482.

19. Songe MM. Pathogenicity and infectivity of Saprolegnia species in Atlantic salmon (Salmo salar L.) and their eggs. 2017.

20. Chukanhom K & Hatai K. Freshwater fungi isolated from eggs of the common carp (Cyprinus carpio) in Thailand. Mycoscience. 2004; 45: 42-48.

21. Ghiasi M, Khosravi A, Soltani M, Sharifpour I, Binaii M, et al. Evaluation of physiological aspects and molecular identification of Saprolegnia isolates from rainbow trout (Oncorhynchus mykiss) and Caspian trout (Salmo trutta caspius) eggs based on RAPD–PCR. isfj. 2014; 22: 82-92.

22. Ke XL, Wang JG, Gu ZM, Li M, Gong XN. Morphological and molecular phylogenetic analysis of two Saprolegnia sp.(Oomycetes) isolated from silver crucian carp and zebra fish. Mycol Res. 2009; 113: 637- 644.

23. Hunjavanit C, Rakmanee C, Kitancharoen N, Hatai K. Freshwater Oomycete Isolated from African Catfish Clarias gariepinus Eggs in Thailand. Aquaculture Science. 2012; 60: 269-276.

24. Melaku H, Lakew M, Alemayehu E, Wubie A, Chane M. Isolation and identification of pathogenic fungus from African Catfish (Clarias gariepinus) eggs and adults in National Fishery and Aquatic Life Research Center Hatchery, Ethiopia. Fish Aqua J. 2017; 8: 1-6.

25. Debnath C, Das BK, Sahoo L. Haematological responses of the Indian major carp Labeo rohita to saprolegniasis. Indian Journal of Fisheries. 2017; 64: 58-62.

26. Van Den Berg AH, McLaggan D, Diéguez-Uribeondo J, Van West P. The impact of the water moulds Saprolegnia diclina and Saprolegnia parasitica on natural ecosystems and the aquaculture industry. Fungal Biology Reviews. 2013; 27: 33-42.

27. Wagner EJ, Oplinger RW, Arndt RE, Forest AM, Bartley M. The safety and effectiveness of various hydrogen peroxide and iodine treatment regimens for rainbow trout egg disinfection. North American Journal of Aquaculture. 2010; 72: 34-42.

28. Brock JA & Bullis R. Disease prevention and control for gametes and embryos of fish and marine shrimp, Reproductive Biotechnology in Finfish Aquaculture. Elsevier. 2001; 137-159.

29. Peck MA, Buckley LJ, O’Bryan LM, Davies EJ, Lapolla, AE. Efficacy of egg surface disinfectants in captive spawning Atlantic cod Gadus morhua L. and haddock Melanogrammus aeglefinus L. Aquaculture Research. 2004; 35: 992-996.

30. Cadirci S. Disinfection of hatching eggs by formaldehyde fumigation–a review. Archiv fur Geflugelkunde. 2009; 73: 116-123.

31. Srivastava S, Sinha R, Roy D. Toxicological effects of malachite green. Aquatic toxicology. 2004; 66: 319-329.

32. Sudova E, Machova J, Svobodova Z, Vesely T. Negative effects of malachite green and possibilities of its replacement in the treatment of fish eggs and fish: a review. Veterinarni Medicina-Praha. 2007; 52: 527.

33. Gieseker C, Serfling S, Reimschuessel, R. Formalin treatment to reduce mortality associated with Saprolegnia parasitica in rainbow trout, Oncorhynchus mykiss. Aquaculture. 2006; 253: 120-129.

34. Khodabandeh S & Abtahi B. Effects of sodium chloride, formalin and iodine on the hatching success of common carp, Cyprinus carpio, eggs. Journal of Applied Ichthyology. 2006; 22: 54-56.

35. Chalupnicki MA, Ketola HG, Starliper CE & Gallagher D. Efficacy and toxicity of iodine disinfection of Atlantic salmon eggs. North American Journal of Aquaculture. 2011; 73: 124-128.

36. Barnes ME, Stephenson H, Gabel M. Use of hydrogen peroxide and formalin treatments during incubation of landlocked fall Chinook salmon eyed eggs. North American Journal of Aquaculture. 2003; 65: 151-154.

37. Small BC & Wolters WR. Hydrogen peroxide treatment during egg incubation improves channel catfish hatching success. North American Journal of Aquaculture. 2003; 65: 314-317.

38. Rasowo J, Okoth OE, Ngugi CC. Effects of formaldehyde, sodium chloride, potassium permanganate and hydrogen peroxide on hatch rate of African catfish Clarias gariepinus eggs. Aquaculture. 2007; 269: 271-277.

39. Gaikowski MP, Rach JJ, Olson JJ, Ramsay RT, Wolgamood, M. Toxicity of hydrogen peroxide treatments to rainbow trout eggs. Journal of Aquatic Animal Health. 1998; 10: 241-251.

40. Kitanchoraroen N, Yamamoto A, Hatai, K. Effects of sodium chloride, hydrogen peroxide and malachite green on fungal infection in rainbow trout eggs. Biocontrol Science. 1998; 3: 113-115.

41. Ghomi MR, Esmaili A, Vossoughi G, Keyvan A, Nazari, R.M. Comparison of ozone, hydrogen peroxide and removal of infected eggs for prevention of fungal infection in sturgeon hatchery. Fisheries science. 2007; 73: 1332.

42. Fry J, Casanova JP, Hamoutene D, Lush L, Walsh A, et al. The impact of egg ozonation on hatching success, larval growth, and survival of Atlantic Cod, Atlantic Salmon, and Rainbow Trout. Journal of aquatic animal health. 2015; 27: 57-64.

43. Rather M, Sharma R, Aklakur M, Ahmad S, Kumar N, et al. Nanotechnology: a novel tool for aquaculture and fisheries development. A prospective mini-review. Fish. Aquacult. J. 2011; 16: 1-15.

44. Huang S, Wang L, Liu L, Hou Y, Li, L. Nanotechnology in agriculture, livestock, and aquaculture in China. A review. Agronomy for Sustainable Development. 2015; 35: 369-400.

45. Luis AIS, Campos EVR, de Oliveira JL, Fraceto LF. Trends in aquaculture sciences: from now to use of nanotechnology for disease control. Reviews in Aquaculture. 2019; 11: 119-132.

46. Khot LR, Sankaran S, Maja JM, Ehsani R, Schuster EW. Applications of nanomaterials in agricultural production and crop protection: a review. Crop protection. 2012; 35: 64-70.

47. Gurianov Y, Nakonechny F, Albo Y, Nisnevitch M. Antibacterial composites of Cuprous Oxide nanoparticles and polyethylene. Int J Mol Sci. 2019; 20: 439.

48. He L, Liu Y, Mustapha A, Lin, M. Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiological Research. 2011; 166: 207-215.

49. Kalatehjari P, Yousefian M, Khalilzadeh MA. Assessment of antifungal effects of copper nanoparticles on the growth of the fungus Saprolegnia sp. on white fish (Rutilus frisii kutum) eggs. The Egyptian Journal of Aquatic Research. 2015; 41: 303-306.

50. Soltani M, Esfandiary M, Sajadi M, Khazraeenia S, Bahonar A, et al. Effect of nanosilver particles on hatchability of rainbow trout (Oncorhynchus mykiss) egg and survival of the produced larvae. IJFS. 2011; 10: 167-178.

51. De Graaf G, Galemoni F, Banzoussi B. Artificial reproduction and fingerling production of the African catfish, Clarias gariepinus (Burchell 1822), in protected and unprotected ponds. Aquaculture Research. 1995; 26: 233-242.

52. Paul Y, Naumann C, Hintz WE. Assessment of intra-specific variability in Saprolegnia parasitica populations of aquaculture facilities in British Columbia, Canada. Dis Aquat Organ. 2018; 128: 235-248.

53. Rasowo J, Okoth OE, Ngugi CC. Effects of formaldehyde, sodium chloride, potassium permanganate and hydrogen peroxide on hatch rate of African catfish Clarias gariepinus eggs. Aquaculture. 2007; 269: 271-277.

54. Policar T, Smyth J, Flanigan M, Kouba A, Kozák P. Sodium chloride as effective antifungal treatment for artificial egg incubation in Austropotamobius pallipes. Knowledge and Management of Aquatic Ecosystems. 2011; 401: 13.

55. Kanhed P, Birla S, Gaikwad S, Gade A, Seabra AB. In vitro antifungal efficacy of copper nanoparticles against selected crop pathogenic fungi. Materials Letters. 2014; 115: 13-17.

56. Saharan V, Sharma G, Yadav M, Choudhary MK, Sharma SS. Synthesis and in vitro antifungal efficacy of Cu–chitosan nanoparticles against pathogenic fungi of tomato. Int J Biol Macromol. 2015; 75: 346-353.

57. He L, Liu Y, Mustapha A, Lin M. Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiol Res. 2011; 166: 207-215.

58. Sawai J & Yoshikawa T. Quantitative evaluation of antifungal activity of metallic oxide powders (MgO, CaO and ZnO) by an indirect conductimetric assay. Journal of Applied Microbiology. 2004; 96: 803- 809.

Received : 23 Aug 2021
Accepted : 27 Sep 2021
Published : 29 Sep 2021
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ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
TEST Journal of Dentistry
ISSN : 1234-5678
Launched : 2014
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
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