Method for binding of water mould spores in aquaculture systems

Abstract

According to an example aspect of the present invention, there is provided a method for binding of water mould spores in aquaculture systems by a cellulose-based material, which is preferably surface modified with molecules having a cationic charge.

Description

METHOD FOR BINDING OF WATER MOULD SPORES IN AQUACULTURE SYSTEMSFIELD

[0001] The present invention relates to a method for binding of water mould spores in aquaculture systems by a cellulose-based material.BACKGROUND

[0002] Saprolegniosis, also known as water mold, is a disease caused by oomycetes from genus Saprolegnia, although other Saprol egniaceae can cause identical symptoms (Pavic et al., 2022). Saprolegniosis can kill fish in all stages of life, from eggs to adult fish. It induces a cotton or wool-like white growth on the fish skin and gills (Yanong, 2003), causing epidermal damage and cellular necrosis (Noga, 2010). Saprolegniosis leads to an impaired ability of osmoregulation, loss of body fluids and finally respiratory failure (vanWest, 2006). The disease is lethal in most cases.

[0003] Saprolegnia spp. is ubiquitous in freshwater ecosystems and is the main genus of water mould responsible for significant fungal infections of freshwater fish and eggs (Pelczar et al., 2008). It causes significant declines in crayfish, fish and amphibian populations (; Pavic et al., 2021) and has been recognized as a threat to biodiversity. Saprolegniosis has been observed in various fish species, amphibians, crustaceans and aquatic insects.

[0004] Saprolegnia spp. is considered responsible for the decline of natural and cultured populations of salmonids, cyprinids, acipensers (Johari et al., 2014), and other aquatic species (van den Berg et al., 2013). Additionally, saprolegniosis occurs frequently in fish hatcheries (Shokouh Saljoghi et al., 2020), on fish farms, and even in hobby fish tanks. Numerous studies have been conducted, but the prevalence of 5. parasitica in the environment or ecological parameters influencing its occurrence remain very limited (Elameen et al., 2021). It is unclear why some farms have frequent outbreaks, whereas others remain disease-free.

[0005] According to some estimates, every freshwater fish is exposed to at least one oomycete during its lifetime, and about 10% of hatched salmon die due to Saprol egnia parasitica (Adel et al., 2020). In aquaculture, Saprolegnia infects especially fresh-water- cultured salmonids such as Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss but also species like eel, perch and catfish (Bruno et al., 2011; Das et al., 2012). Saprolegniosis causes mortality and presents a great threat to animal well-being (Magray et al., 2019). The initial cause of saprolegniosis outbreak is still unknown, but stressors, such as poor water quality, handling, and high fish density, have been associated with disease outbreaks (Zahran & Risha, 2013).

[0006] Various treatments have been used to control saprolegniosis, such as hydrogen peroxide (Ali et al., 2019), formalin (Bly et al., 1996), Bronopol (Pyceze), NaCl, acetic acid, povidone iodine (Fuangsawat et al., 2011), ozone, UV light (Rahkonen & Koski, 2002), KMnO4(Sherif & Abdel-Hakim, 2016), CIO2 (Prasatporn et al., 2010), and triclosan (Kumar et al., 2020). However, they all are less efficient than malachite green (Bruno et al., 2011) which was banned in 2002 (due to toxicity,). Many include safety concerns, causticity can cause tissue damage, or their application in large volumes of water is impractical (Oono et al., 2008). The currently most applied chemicals against Saprolegnia spp. are formaldehyde, peracetic acid and hydrogen peroxide (Adel et al., 2020). Multiple chemicals have been tested to control saprolegniosis and other parasites (Buchmann, 2022), but their harmful effects on farming personnel or the environment often prevent their wider use (Kumar et al., 2020).

[0007] Malachite green was long considered the most effective treatment (Srivastava et al., 2004). It has been used routinely since the 1930s due to its efficiency against fungal infections. Malachite green was banned in edible fish in the United States, Europe (Legislative Decree no. 119 / 92, Forneris et al., 2003), and worldwide in 2002 due to its carcinogenic and toxic effects and teratogenic and mutagenic properties (Kumar et al., 2020). This has forced farmers to use alternative disinfection methods against saprolegniosis (Sandoval-Sierra et al., 2014). Currently, no sufficiently effective treatment has been found, which has for its part led to the spread of saprolegniosis (Kumar et al., 2020) and struggle for many fish farmers (Derevnina et al., 2016).

[0008] Formalin, an aqueous solution of 37% formaldehyde, is commonly used. Formalin is currently registered as a disinfectant and / or parasiticide for use in aquaculture(Leal et al., 2018). Formalin is widely used but is not effective enough to control Saprolegnia infections in fish, fish eggs (Bruno et al., 2011), and larvae. However, there are concerns about its effects on the environment and the handling personnel. Formalin has acute effects on aquatic ecosystems, teratogenic and immunosuppressive for fish if used repeatedly.

[0009] CH717692A1 discloses a cellulose-based filter for separating bacteria or viruses from water, such as drinking water. This patent publication, however, does not disclose use of the filter material for treatment of water in a recirculating aquatic system nor is farming of aquatic organisms mentioned. Further, the filtering of mould spores is not disclosed.

[0010] CN110199996 A discloses a method for preparing an aquaculture bactericide, wherein chitosan is modified with 2,3-propyltrimethylammonium chloride. This patent publication, however, does not disclose a cellulose-based carrier nor separation of mould spores from water.

[0011] EP3294674B1 discloses the use of cationic nanofibrillar cellulose as a w'ater treatment chemical or an additive to remove ions from the water. The nanofibrillar cellulose is cationized using epoxypropyltrimethyl ammonium chloride. This patent publication, however, does not disclose filtering or removal of mould spores.

[0012] In aquaculture, there is an urgent need for a new and effective treatment agent without harmful side-effects to the reared species, environment or farming personnel. In the future, connections among different water quality parameters with saprolegniosis occurrences need to be systematically and scientifically studied. For fish farmers, this could act as warning signs for saprolegniosis and as a signal for starting a treatment. It is of high importance to find an effective and non-toxic treatment agent, and research efforts should be made to achieve this goal.SUMMARY OF THE INVENTION

[0013] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0014] According to an aspect of the present invention, there is provided a method for binding of water mould spores in aquaculture systems by a cellulose-based material.

[0015] According to a further aspect of the present invention, the cellulose-based material is surface modified with molecules having a cationic charge.

[0016] These and other aspects, together with the advantages thereof over known solutions are achieved by the present invention, as hereinafter described and claimed.

[0017] The method of the present invention is mainly characterized by what is stated in the characterizing part of claim 1.

[0018] Considerable advantages are obtained by means of the invention. Water mold caused by Saprolegnia parasitica is a serious freshwater fish disease that almost invariably results in the death of the infected individual. This can lead to significant economic losses at aquaculture facilities or cause the depletion of valuable fish stocks in water systems. Water mold occurs all over the world in freshwater systems in nature and in aquaculture facilities. The connection between disease cases and water quality factors and other conditions is still relatively poorly known. Water mold infects fish individuals via spores by attaching to the fish's skin, most commonly fins or snout. By removing or reducing water mold spores from the system, water mold infections and fish deaths caused by it can be reduced or completely prevented. This can have significant economic implications in fish production. In addition, viscose fabric for the support material is readily available.

[0019] Next, the present technology will be described more closely with reference to certain embodiments.EMBODIMENTS

[0020] The present technology provides means for binding of water mould spores, in particular of the species Saprolegnia parasitica, in aquaculture systems with cellulose- based materials, which have been surface modified with molecules having a cationic charge.

[0021] FIGURE 1 is a chart showing the amount of Saprolegnia parasitica spores in water per 100 mL during time (measurement weeks). Sample 1 is a reference woven cottonfabric, sample 2 is a reference viscose non-woven fabric, sample 3 is a GTAC-cationized woven cotton fabric and sample 4 is a chitosan-modified non-woven fabric.

[0022] FIGURE 2 is a chart showing the amount of Saprolegnia. spores in fabric per 100 mL during time (measurement weeks). Sample 1 is a reference woven cotton fabric, sample 2 is a reference viscose non-woven fabric, sample 3 is a GTAC-cationized woven cotton fabric and sample 4 is a chitosan-modified non-woven fabric.

[0023] The present invention is based on applying a cellulose-based material, which is surface modified with molecules having ability to bind water mould spores, to an aquaculture system.

[0024] More precisely, one aspect of the present invention is a method for binding of water mould spores in aquaculture systems by a cellulose-based material, wherein the method comprises introducing a native plant-based cellulose material or a regenerated cellulose material to an aquaculture system, wherein the cellulose material is surface modified with molecules having a cationic charge, and thereby binding water mould spores from the aquaculture sy stem.

[0025] According to one embodiment of the present invention, the water mould spores are of the species Saprolegnia, in particular Saprolegnia parasitica.

[0026] According to one embodiment of the present invention, the surface modification comprises providing the cationic charge to a surface of the cellulose material by for example attachment of glycidyl trimethyl ammonium groups or by chitosan adsorption.

[0027] According to one embodiment of the present invention, the method comprises trapping anionic water mould spores mainly to the cationic groups of the surface modified cellulose material.

[0028] According to one embodiment of the present invention, a glycidyl trimethyl ammonium chloride (GTAC)-cationized cellulose material and / or a chitosan-modified cellulose material binds more Saprolegnia spores than a cellulose material without any cationic modification.

[0029] According to one embodiment of the present invention, the method comprises introducing the surface modified cellulose material into an aquaculture system for farming aquatic organisms, such as into a fish farming tank and / or pond.

[0030] According to one embodiment of the present invention, the aquaculture system is a recirculating aquaculture system (RAS).

[0031] According to one embodiment of the present invention, the method comprises selecting the cellulose material from cellulose non-wovens, cellulose fabrics, viscose fabric, lyocell fabrics, modal fabrics, cotton, hemp fabrics, aerogels and particles trapped within an inert permeable material.

[0032] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed.

[0033] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it wall be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0034] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", that is, a singular form, throughout this document does not exclude a plurality.INDUSTRIAL APPLICABILITY

[0035] The present technology is applicable in aquaculture systems, such as in fish farming systems, for binding of water mould spores.EXAMPLESExperimental set up:® landlocked salmon Salmo salar m. sebago• 4x2 tanks (depth 70 cm; 8,9 m3; 12,7 m2), flow-through systems• 200 fish, average size of 260 g, biomass per tank 52 kg; fish density 4,09 kg / m2• Water flow to tanks 3,5 L / s (= 12,6 m3 / h)® Change of fabric once a week• Chemical cationization of cellulosic fabric was conducted by immersing the fabric in a reaction mixture of glycidyl trimethyl ammonium chloride (GTAC), isopropanol, water, and NaOH at 60 °C for 6 hours. After this, the reaction mixture was neutralized with 10% HC1 to pH 7-8, followed by extensive washing of the fabrics with water.® Regenerated cellulose (viscose) non-woven was modified by using a two-step process. First chitosan was dissolved into 0.1 M acetic acid buffer at pH 5. Then it was freely allowed to adsorb on the surface of viscose fabric for a period of 1 hour. After the adsorption, the fabric was rinsed with pure buffer solution for 1 hour.Results of the experiment are presented in Table 1 (fish), Figure 1 (water) and Figure 2 (fabric).Table 1. Saprolegnia copies per g fish on observation week 3 (weeks I&2 no observations)Week 3 Saprolegnia copies per gFabric Average Standard deviation 1. reference woven cotton fabric 11378 123632. reference viscose non-woven fabric 2110 29853. GTAC-cationized woven cotton fabric 12576702 2 783 7474. chitosan-modified non-woven fabric 67018 30627CITATION LISTPatent literatureCH717692A1CN110199996AEP3294674B1Non-patent literature:Adel, M., Dadar, M., Zorriehzahra, M. J., Elahi, R. & Stadtlander, T. (2020) Antifungal activity and chemical composition of Iranian medicinal herbs against fish pathogenic fungus, Saprolegnia parasitica. Iranian Journal of Fisheries Sciences, 19, 3239-3254. https: / / doi.org / 10.22092 / ijfs.2020.122970Ali, S. E., Ganiil, A. A. A., Skaar, I., 0, E. & Charo-Karisa, H. (2019) Efficacy and safety of boric acid as a preventive treatment against Saprolegnia infection in Nile tilapia Oreochromis niloticus). Scientific Reports, 9, 18013. https: / / doi.org / 10.1038 / s41598- 019-54534-yBly, J. E., Quiniou, S. M.-A., Lawson, L. A. & Clem, L. W. (1996) Therapeutic and prophylactic measures for winter saprolegniosis in channel catfish. Diseases of Aquatic Organisms, 24, 25-33. https: / / doi.org / 10.3354 / dao024025Bruno, D., vanWest, P. & Beakes, G. (2011) Saprolegnia and other Oornycetes. In: Woo, P., Bruno, D. (Eds.) Fish diseases and disorders, viral, bacterial and fungal infections, 2nd edition, Wallingford, UK: CABI: Wallingford, pp. 669-720.Buchmann, K. (2022) Control of parasitic diseases in aquaculture. Parasitology, 149, 1985-1997. https: / / doi.org / 10.1017 / S0031182022001093Chukanhom, K. & Hatai, K. (2004) Freshwater fungi isolated from eggs of the common carp (Cyprinus carpio) in Thailand. Mycoscience, 45, 42-48.https: / / doi. org / 10.1007 / s 10267-003 -0153 -9Das, S. K., Murmu, K., Das, A., Shakuntala, I., Das, R. K., Ngachan, S. V. et al. (2012) Studies on the identification and control of pathogen Saprol egnia in selected Indian major carp fingerlings at mid hill altitude. Journal of Environmental Biology, 33, 545-549.Derevnina, L., Petre, B, Kellner, R., Dagdas, Y. F., Sarowar, M. N., Giannakopoulou, A. et al. (2016) Emerging oomycete threats to plants and animals. Philosophical Transactions of the Royal Society B, 371, 20150459. http: / / doi.org / 10.1098 / rstb.2015.0459 Elameen, A., Stueland, S., Kristensen, R., Fristad, R. F., Vralstad, T. & Skaar, I. (2021) Genetic analyses of Saprolegnia strains isolated from salmonid fish of different geographic origin document the connection between pathogenicity and molecular diversity. Journal of Fungi, 7, 713. https: / / doi.org / 10.3390 / jof7090713Fernandes, C., Lalitha, V. & Rao, K. (1991) Enhancing effect of malachite green on the development of hepatic pre-neoplastic lesions induced by N-nitrosodiethylamine in rats. Carcinogenesis, 12, 839-845. https: / / doi.org / 10.1093 / carcin / 12.5.839Femandez-Beneitez, MJ., Ortiz-Santaliestra, M. E., Lizana, M. & Dieguez-Uribeondo, J. (2008) Saprolegnia diclina: another species responsible for the emergent disease ‘Saprolegnia infections' in amphibians. FEMS Microbiology Letters, 279, 23-29. https: / / doi.org / 10.1111 / j.1574-6968.2007.01002.xForneris, G., Bellardi, S., Palmegiano, G. B., Saroglia, M., Sicuro, B., Gasco, L. et al.(2003) The use of ozone in trout hatchery to reduce saprolegniasis incidence.Aquaculture, 221, 157-166. https: / / doi.org / 10.1016 / S0044-8486(02)00518-5 Fuangsawat, W., Abking, N. & Lawhavinit, O. (2011) Sensitivity comparison of pathogenic aquatic fungal hyphae to sodium chloride, hydrogen peroxide, acetic acid and povidone iodine. Kasetsart Journal (Natural Science), 45, 84-89.Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S. L., et al. (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature, 484(7393), 186-194. https: / / doi.org / 10.1038 / nature10947Kumar, S., Mandal, R. S., Bulone, V. & Srivastava, V. (2020) Identification of growth inhibitors of the fish pathogen Saprolegnia parasitica using in silico subtractiveproteomics, computational modeling, and biochemical validation. Frontiers in Microbiology, 11, 571093. https: / / doi.org / 10.3389 / fmicb.2020.571093Leal, IF., Neves, M. G. P. M. S., Santos, E. B. H. & Esteves, V I. (2018 ) Use of formalin in intensive aquaculture: properties, application and effects on fish and water quality. Reviews in Aquaculture, 10, 281-295. https: / / doi.org / 10.1111 / raq.12160 Magray, A.R., Lone, S.A., Ganai, B.A., Ahmad, F., Dar, G.J., Dar, J.S. et al. (2019) Comprehensive, classical and molecular characterization methods of Saprolegnia (Oomycota; Straminipila) an important fungal pathogen of fish. Fungal Biology Reviews, 33, 166-179. https: / / doi. Org / 10.1016 / j.fbr.2018.12.001Noga, E. J. (2010) Fish disease: diagnosis and treatment. Iowa, USA: John Wiley & Sons. Oono, H., Hatai, K., Aikawa, H.& Hara, H. (2008) The use of bronopol to control fungal infections inAyu eggs. Aquaculture Science, 56, 9-12.https: / / ci.nii.ac.jp / naid / 10026138916 / en / Pavic, D., Grbin, D., Hudina, S., Zmrzljak, U. P., Miljanovic, A., Kosir, R. et al. (2022) Tracing the oomycete pathogen Saprolegnia parasitica in aquaculture and the environment. Scientific Reports, 12, 16646. https: / / doi.org / 10.1038 / s41598-022-16553- 0Pelczar, M. J., Chan, E. C. S. & Krieg, N. R. (2008) Microbiology, Sth editions. New Delhi, India: Tata McGraw Hill Publishing Company Ltd.Prasatporn, B., Pithai, K., Chutima, FL, Horiuchi, E,, Wada, S. & Hatai, K. (2010) The in vitro antifungal effects of chlorine dioxide on water molds. Aquaculture Science, 58, 219-224. http s: / / doi. org / 10.11233 / aquacul tare sci.58.219Rahkonen, R. & Koski, P. (2002) Post malachite green: alternative strategies for fungal infections and white spot disease. Bulletin of the European Association of Fish Pathologists, 22, 152-157.Sandoval -Sierr, J. V., Martin, M. P. & Dieguez-Uribeondo, J. (2014) Species identification in the genus Saprolegnia (Oomycetes): defining DNA-based molecular operational taxonomic units. Fungal Biology, 118, 559-578.https: / / doi.org / 10.1016 / j.funbio.2013.10.005Sherif, A. H. & Abdel -Hakim, S. (2016) Treatment trails of saprolegniosis in Oreochromis niloticus. Alexandria Journal of Veterinary Sciences, 49, 99-104. https: / / doi.org / 10.5455 / ajvs.226020Shokouh Saljoghi, Z., Farhadian, O., Ramezanian, N. & Mehraban Sangatash, M. (2020) Synthesis and characterization of novel compounds and determining their antifungalI Iproperties against rainbow trout pathogen, Saprolegnia sp. in vitro. Iranian Journal of Fisheries Sciences, 19, 1396-1414. https: / / doi.org / 10.22092 / ijfs.2019.120048.0 Schnick, R. A. (1988) The impetus to register new therapeutants for aquaculture. The Progressive Fish-Culturist, 50, 190-196. https: / / doi.org / 10.1577 / 1548- 8640(1988)050(0190:titrnt)2.3. co;2Yanong, R. P. (2003) Fungal diseases offish. 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Claims

CLAIMS:

1. A method for binding of water mould spores in aquaculture systems by a cellulose-based material, characterized by introducing a native plant-based cellulose material or a regenerated cellulose material to an aquaculture system, wherein the cellulose material is surface modified with molecules having a cationic charge, and thereby binding water mould spores from the aquaculture system.

2. The method according to claim 1, characterized in that the water mould spores are of the species Saprolegnia, in particular Saprolegnia parasitica.

3. The method according to claim 1 or 2, characterized in that the surface modification comprises providing the cationic charge to a surface of the cellulose material by for example attachment of glycidyl trimethyl ammonium groups or by chitosan adsorption.

4. The method according to claim 3, characterized in trapping anionic water mould spores mainly to the cationic groups of the surface modified cellulose material.

5. The method according to claim 3 or 4, characterized in that a glycidyl trimethyl ammonium chloride (GTAC)-cationized cellulose material and / or a chitosan-modified cellulose material binds more Saprolegnia spores than a cellulose material without any cationic modification.

6. The method according to any of the preceding claims, characterized by introducing the surface modified cellulose material into an aquaculture system for farming aquatic organisms, such as into a fish farming tank and / or pond.

7. The method according to any of the preceding claims, characterized in that the aquaculture system is a recirculating aquaculture system (RAS).

8. The method according to any of the preceding claims, characterized by selecting the cellulose material from cellulose non-wovens, cellulose fabrics, viscose fabric, lyocell fabrics, modal fabrics, cotton, hemp fabrics, aerogels and particles trapped within an inert permeable material.

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