Environmental assessment of the use of biologically active substances of forage plants in fish farming

. This article focuses on the environmental assessment of the use of biologically active substances (BAS) of forage plants in fish farming. The main attention is given to the study of the degree of microbial contamination of plant components included in the feed. The objective of the study was to identify the spectrum of contamination of grower feeds for rainbow trout with micromycetes. Due to an environmental assessment of feed samples containing forage plants (soy bean, rapeseed, wheat) or their components (soybean flour, sunflower meal), representatives of three genera of micromycetes, such as Aspergillus, Fusarium, and Rhizopus, belonging to the kingdom of Fungi, divisions Ascomycota and Zygomycota; subclasses Eurotiomycetidae, Sordariomycetidae, and Mucorineae; orders Eurotiales, Hypocreales, Mucorales, and families Trichocomaceae, Nectriaceae, and Rhizopodaceae, were found. The detrimental effect of myxomycetes on the bodies of fish has been detected.


Introduction
The increase in fish farming is associated with the quality of grower feeds, which include a sufficient amount of biologically active substances (BAS) of plant origin.The following elements are included in BAS: vitamins, phytoncides, hormones, and enzymes, as well as a group of macro-and micro-elements.Among the plant BAS, bio-antioxidants of flavonoid origin are also distinguished.The flavonoid dihydroquercetin extracted from larch is characterized by a wide spectrum of effects; it is an integral component in the synthesis of vitamin P, and its role in reducing the fragility and permeability of capillaries is widely known [4].The overwhelming number of important biologically active substances are not synthesized in the body of fish and are ingested only with feed in the form of premixes [5].It has been experimentally proven that the effect of a lack of feed causes not only a disturbance of enzyme synthesis but also a nonreversible metabolic imbalance, contributing to the development of physical illnesses and a decrease in the resistance of fish to damaging environmental factors [11].
The total proportion of plant components in fish diets can reach about 90% and include compounds rich in fat, starch, or protein.The main sources of plant components are soybeans, rye, ground oat, and barley.In some cases, wheat bran, corn, and waste from oil production or meal-soy, linseed, or sunflower-are used in the formation of fish feeds.For example, 1 kg of grower feed may contain fiber up to 144 g/kg, proteins up to 430 g/kg, and fats up to 140 g/kg.Wheat bran is an essential source of vitamins E and B; coniferous flour includes carotene, phylloquinone, ascorbic acid, provitamins of group D, tocopherol, and riboflavin [6].
Nonetheless, despite the significant component role, the use of BAS of forage plants in fish farming is directly related to the severe challenge of increased contamination of plant raw materials with mycotoxins.According to the data [2], low-quality plant raw materials account for around 85% of cereal major diseases, with approximately 80% represented by toxigenic microscopic fungi, including Aspergillus, Alterna, Fusarium, and others.The most well-known of these representatives is aflatoxin, which by nature cannot be destroyed by heat treatment.This compound can also affect the liver, reduce the activity of protein biosynthesis, lower the level of prothrombin, and adversely affect the immune system of fish and reproductive ability [9].If the concentration of aflatoxin-affected food in rainbow trout is 20 mg/t, it may result in the formation of hepatocellular carcinoma [11].Non-compliance with the transportation, processing, storage, and transportation technology of plant raw materials for the production of grower feed may cause the accumulation of ecotoxicants of natural origin and aggravate the problem.In the framework of the above, one should not ignore the anti-nutritional factors of plant raw materials, primarily associated with the biosynthesis of compounds by plants, which are a factor in the suppression of a number of biological processes.These include erucic acid, an inhibitor of soybean meal trypsin, as well as lysine with carbohydrates resistant to the action of digestive enzymes, resulting from the Maillard reaction when heated [10].
In light of the foregoing, an important consideration for the selection of forage plants as part of compound feeds for aquaculture facilities is not only their utility as a source of BAS, but also their environmental safety.Meanwhile, ecological safety of feed is defined as the absence of any negative environmental impact during manufacturing and consumption.
The objective of the study was to identify the spectrum of contamination of grower feeds for rainbow trout with micromycetes.
The environmental assessment was performed under GOST R 51426-2016 Microbiology.Feedstuffs, compound feeds, and feed raw materials.General guidance for the preparation of dilutions for microbiological examination and the microbiology of food and animal feeding stuffs.Methods for the detection and colony count of yeasts and molds.To determine yeastlike and mold-like fungi, a 1g feed sample was used, selected from an average sample (the single-step selection of feed for the sample).
Five samples of compound feed were chosen for the study, containing vegetable components specified in Table 1 in addition to basic and vitamin-mineral complexes.All samples of the studied compound feed contained wheat; a small proportion of soy flour, non-GMO eco-soybean, and rapeseed were also found.Morphological signs of isolated micromycetes were assessed on intravital specimens by the presence of phialides, microconidia, conidia, metula, and conidiophores (fig.1).Due to an environmental assessment of feed samples containing forage plants (soy bean, rapeseed, wheat) or their components (soybean flour, sunflower meal), representatives of three genera of micromycetes, such as Aspergillus, Fusarium, and Rhizopus, belonging to the kingdom of Fungi, divisions Ascomycota and Zygomycota; subclasses Eurotiomycetidae, Sordariomycetidae, and Mucorineae; orders Eurotiales, Hypocreales, Mucorales, and families Trichocomaceae, Nectriaceae, and Rhizopodaceae, were found.
Aspergillus -is the cause of mycotoxicosis associated with the action of furocoumarins, which include the aflatoxin family: B1, B2, G1, G2.Aspergillus is found more often on rye and wheat; aflatoxins are thermally stable and do not destroy during feed processing.Aflatoxins act on metabolic processes and disrupt the functions of the immune system in the body of fish [9].
Fusarium -is the reason for the onset of many diseases of agricultural crops.Representatives of the genus are found in soil and on plants as endophytes, saprophytes, and parasites; type of parasitism: facultative saprotrophy.Fusarium is a producer of deoxynivalenol (DON), which is metabolized into DON-3-sulfate in the trout body.DON-3sulfate is much less toxic than DON, which explains the failure of clinical signs in fish treated with DON [9].During ichthyopathological examination of trout aged 2+, signs of mycotoxicosis were registered, probably caused by the existence of trichothecenes, which produce fungi of the genus Fusarium.Discoloration (paleness), change in consistency (sagginess) and volume of parenchymal organs (Fig. 2a), and hemorrhages in the intestine (Fig. 2b, 2c) were found upon autopsy of sick fish).Hematological examination showed RBC hemolysis.In some cases, there was a decrease in the length and an increase in the width of the fish's body, as well as swelling of the anal papilla (Fig. 3).It is understood that when feeds are contaminated with DON, appetite suppression appears, which may be a protective mechanism to reduce the effects of DON on the trout's body, hence reducing the potential adverse effects of DON.If DON enters the body, the activity of trypsin greatly reduces, which has a direct effect on the level of insulin, which ultimately affects the absorption of amino acids.DON affects the neuroendocrine regulator of satiety and food intake, pituitary adenylate cyclase-activating polypeptide (PACAP).This polypeptide acts as a specific neurotransmitter that is synthesized by the organs of the digestive tract and may have diagnostic value [8].Decreased appetite and increased expression of PACAP genes, associated with the ingestion of deoxynivalenol (DON), act as a defense mechanism to reduce the ingestion of mycotoxin-infected feed.According to papers describing the transformation of deoxynivalenol [7], deoxynivalenol (DON) is transformed into deoxynivalenol-3-sulfate (DON-3) by microorganisms.This process results in minimally toxic effects of DON on the gastrointestinal tract and largely explains why fish that were fed mycotoxin-infected fish did not exhibit symptoms of mycotoxicosis.
Rhizopus lives in the soil and on plants.It causes diseases of agricultural crops: wheat, corn, etc. Rhizopus belongs to toxigenic fungi.
The safety assessment of production feeds for aquaculture facilities indicates that plant components are a crucial source of BAS for grower feeds for trout.These BAS have a positive impact on fish growth and development in cage keeping conditions, as well as improving productivity and product quality.However, the presence of micromycetes of the genera Aspergillus, Fusarium, and Rhizopus in grower feeds may indicate a violation of technological standards in the production or storage of feed.This can trigger various diseases in aquaculture facilities and reduce the quality of products.The isolated micromycetes are able to form mycotoxins with a whole range of adverse effects.Controlling the presence of micromycetes and, if required, determining the concentration of mycotoxins in compound feeds are crucial in this regard.Therefore, if used correctly and under close supervision, the biologically active components of forage plants can be both safe and successful when used in fish farming.Comprehensive environmental studies are necessary to assess the effects of mycotoxin producers on aquatic creatures and ecosystems overall, especially in light of the growing contamination of forage plants by micromycetes.

Fig. 1 .
Fig. 1.Morphological signs of isolated micromycetes: a. phialides, b. microconidia, c. conidia, d. metula, e. conidiophores.The obtained data underwent statistical processing according to generally accepted techniques[1] using the Microsoft Excel 2003 spreadsheet software.Due to an environmental assessment of feed samples containing forage plants (soy bean, rapeseed, wheat) or their components (soybean flour, sunflower meal), representatives of three genera of micromycetes, such as Aspergillus, Fusarium, and Rhizopus, belonging to the kingdom of Fungi, divisions Ascomycota and Zygomycota; subclasses Eurotiomycetidae, Sordariomycetidae, and Mucorineae; orders Eurotiales, Hypocreales, Mucorales, and families Trichocomaceae, Nectriaceae, and Rhizopodaceae, were found.Aspergillus -is the cause of mycotoxicosis associated with the action of furocoumarins, which include the aflatoxin family: B1, B2, G1, G2.Aspergillus is found more often on rye and wheat; aflatoxins are thermally stable and do not destroy during feed processing.Aflatoxins act on metabolic processes and disrupt the functions of the immune system in the body of fish[9].Fusarium -is the reason for the onset of many diseases of agricultural crops.Representatives of the genus are found in soil and on plants as endophytes, saprophytes, and parasites; type of parasitism: facultative saprotrophy.Fusarium is a producer of deoxynivalenol (DON), which is metabolized into DON-3-sulfate in the trout body.DON-3sulfate is much less toxic than DON, which explains the failure of clinical signs in fish treated withDON [9].During ichthyopathological examination of trout aged 2+, signs of mycotoxicosis were registered, probably caused by the existence of trichothecenes, which produce fungi of the genus Fusarium.Discoloration (paleness), change in consistency (sagginess) and volume of parenchymal organs (Fig.2a), and hemorrhages in the intestine (Fig.2b, 2c) were found upon autopsy of sick fish).Hematological examination showed RBC hemolysis.In some cases, there was a decrease in the length and an increase in the width of the fish's body, as well as swelling of the anal papilla (Fig.3).

,Fig. 2 .
Fig. 2. Similar changes in organs and tissues of trout described in the paper by Mikulich Ye.L. [3] (a, b) and revealed during autopsy (c).

Table 1 .
Plant components of the studied samples of grower feed for rainbow trout.