Qualitative analysis, antioxidant properties of azolla and duckweed against chemo-stress in carp fish exposed to CuSO4

Introduction

Rapid industrialization and urbanization have led to heavy metal pollution in freshwater ecosystems, contaminating aquatic food supplies and increasing metal flux from terrestrial sources (1). The presence of heavy metal residue in vegetables grown in polluted areas, as well as fish and shellfish taken from polluted natural sources, raises concerns about health of human (2,3). Many heavy metals (Cu, Zn, Se, Co, Cr and Mn, are required for basic cellular functions), found in freshwater are undesirable and unimportant to aquatic ecosystems. Heavy metals, on the contrary hand, that are generally essential for fish and other aquatic species to grow normally are frequently in excess of what these aquatic organisms require and end up being harmful to them (4,5). As agricultural output has increased, so has the number of lakes and rivers exposed to potentially hazardous toxins such as copper-containing herbicides. Small wastewater flows can easily convey toxic pollutants into rivers and lakes. Copper sulphate (CuSO₄) is employed commonly as a pesticide in agriculture to combat protozoal and fungal infestation, Copper ions were found at quantities within the permissible limit in a number of freshwater aquatic ecosystems including ground waters (6,7). Copper sulphate is frequently utilized in aquaculture as a potent medication for preventing and treating various ectoparasitic and bacterial diseases (8). Fungi, protozoa, and trematodes are exposure alone. When exposed to copper ions, Salmonella spp., Pasteurella spp., Vibrio spp., and other bacteria all grown adversely (9). Copper treatment and repeated exposure can significantly increase the amount of this hazardous element in aquaculture ponds (10), lasting harmful for at least 50 hours after one treatment and potentially impairing fish development (11,12). Furthermore, in a catfish farm in Vietnam, the fish have been proven to collect copper (13,14). Azolla is a watery floating plant that produces a significant amount of protein and biomass. It can be used directly in fish feed or as a dietary component for another protein source (15). Azolla has a greater crude protein level 13 to 30% and an essential amino acid (EAA) makeup that is high in lysine when compared with other green crop varieties and aquatic macrophytes (16), beside phyto-constitutuents such as flavonoids, phenols, alkaloids and saponins (17). Duckweed is a member of the floater family. There are now 40 species classified as belonging to the four genera Lemna, Spirodela, Wolfilla, and Wolffiella, the most well-known species of Lemna is Lemna minor. It can be generated in large quantities at a low cost and is far more cost effective than other plant protein alternatives. Duckweed has recently gained popularity as a high-protein (40-45% dry weight) fish food. Duckweed protein is more akin to animal protein in this regard, as it includes more of the necessary amino acids lysine and methionine than other plant proteins, also Duckweed has high levels of flavonoids, minerals, vitamins and fatty acids (PUFAs) (18,19). Blood tests were administered to fish exposed to various water pollutants and toxicants, such as metals, chemicals, pesticides, and industrial effluents, as a sensitive indicator for stress (20). Knowing normal and pathological processes, as well as toxic effects, in fish is now heavily reliant on blood parameter monitoring. Fish blood is utilized in toxicology research and environmental monitoring as a valuable indicator of physiological and pathological changes, aiding scientists in understanding the harm caused by contaminants. Biochemical indicators such as glucose and lipid profile are widely used to assess the toxicity of environmental pollutants (21). Although there have been some histological investigations on the effects of various pollutants on fish kidneys, little is known regarding the influence of copper on the histopathology of exposed fish (22,23).

To identify the effects of Azolla and Duckweed as anti-oxidant aquatic plant, blood biochemical parameters of common carp exposed to copper sulfate were investigated in the current study.

 Materials and methods

Fertilizer propagation

Azolla species are cultivated in a mildly shaded pond using dechlorinated tap water. Sheep excrement is used to make fertilizer, and the biomass is extracted, dried, then kept in the plastic bag (24).

Cultivation of Duckweed

The concrete ponds of 200 m²/ (20 mO10 m) ready for Duckweed cultivation. Following a thorough cleaning of the pond's bottom, ground water (50 cm) was added. At a rate of 1.052 kg per square meter, cattle dung, bird feces, mustard oil cake (1:1:1) were employed. Duckweed was created using organic manures, chosen based on the results of exterior concrete tanks. The cultivation conditions for (Lemna spp.) that were created during the tank trials were adopted in the pond. Eventually, Mosul University's agriculture institution produced new Duckweed in cement tanks at a rate of 1 kilogram per pond. It just partially covered the pond. Harvesting began once the plant had completely covered the pond's surface (25).

Preparation of plants extracts

The Fatty acids were isolated from Azolla and Duckweed using (100gm) of the ground sample placed in a thimble soaked in a Soxhlet apparatus with 500 ml petroleum ether 60-80ºC for 48 hours until the color of solvent became clear, using a rotary evaporator, the fatty extract was then packed into sterile bottle and placed in a 2-4ºC refrigerator. The oil isolated from Azolla end Duckweed was diagnosed by HPLC technique. Phenolic compounds were obtained from the remaining tissues, were taken and dried to get rid of petroleum ether and the ground sample of the plants (each on at time) placed in a thimble, soaked in a Soxhlet apparatus with 500 ml ethanol (90%⁾ for 48 hours until it became clear. The alcoholic crude extract was obtained after drying through rotary evaporator, the crud extract was kept in an amber vial and placed in the refrigerator, phenolic compounds were identified using HPLC technique (26)^.

Experimental design

In Mosul University's Fisheries Faculty's College of Agriculture conducted the study. The 56-day test was carried out in a round plastic tank with a pH of 8.1±0.3 and a temperature of 22°C. A total of 125 common carps weighing 71.3±0.5 gm was assigned by random to one of five experiment groups: Group the negative control (untreated) first. Positive control in the second group 0.5 mg/L copper sulfate, CuSO₄. Azolla treatment in Group 3 with a CuSO₄ dose of 0.5 mg/liter. Fourth Group CuSO₄ 0.5 mg/L treatment with Duckweed plant water. CuSO₄ from the Fifth Group and a combination of Azolla and Duckweed. The entire therapy period is 56 days. The entire fish population in each treatment was evaluated for gross clinical symptoms.

Sampling

Blood samples were centrifuged at 3000 rpm for 10 minutes on a Vitros- System –Chemistry- 350 after serum extraction and stored at -20°C for assay preparation.

Analysis of Biochemical parameters

A veterinary biochemistry analyzer was used to test blood enzymes such as total cholesterol (TC), triglyceride (TG), globulin, albumin, and total protein (VS2 Abaxis, USA).

Histopathological analysis

Five groups of 125 carp fish were created at random. After the 56-day treatment, the fish were slain so that samples of their intestines and gills could be obtained for rotational histopathological investigation (27).

Statistical investigation

 SPSS version was used to examine the data, using one- way ANOVA and independent t-test to generate cross –tabs and conclusions. The impacts of continuous variables were depicted using mean and standard error.

Results

Qualitative analysis

In the current study fatty acids were diagnosed using HPLC technique, depending on the time of their purity in the separation rod, which in turn depends on the length of the hydrocarbon chain of fatty acid and degree of saturation of fatty acid (Table 1 and Figure 1). The oil extract of both Azolla and Duckweed plants had lowest contents of saturated fatty acids, and the highest of poly unsaturated fatty acids, and monounsaturated fatty acid. The phenolic and flavonoids were also identified by HPLC technique by comparing the retention time of standard compounds (Table 2 and Figure 2).

Table 1: Fatty acids and their percentage in Azolla and Duckweed oil extract

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Figure 1: HPLC chromatogram of fatty acids extraction in a): Azolla oil, b): Duckweed oil

Table 2: Compound found in Azolla and Duckweed (alcoholic crude extract)

Figure 2: Overlaid HPLC phenolic profile of (a): Azolla (diagnostic compounds: Gallic acid 2.01, Chlorogenic acid 3.16, Caffeic acid 4.81, Ferulic acid 6.08, Ellagic acid 7.50, P-coumaric acids 8.21, Quercetin, 11.40) and (b): Duckweed (Gallic acid 2.01, Chlorogenic acid 3. 18, Caffeic acid 4.81, Ferulic acid 6.06, Rutin, 9.89)

Biochemical test

Table 3 shows the concentrations of albumin, total protein, triglycerides, total cholesterol, and globulin in the study groups. The results showed that group 2 (Copper sulfate) had a considerably higher total cholesterol of 6.174±0.304, followed by group 5 (copper sulfate with a mixture of Azolla and Duckweed) and group 4 (copper sulfate with water Duckweed plant) of 3.644±1.618 and 2.936±0.0882, respectively, while group 3 (copper sulfate with Azolla) had reduced total cholesterol levels of 2.726±0.223 compared to controls at 2.884±0.239. Triglyceride levels were greater in group 2 (copper sulfate) 2.772±0.345, followed by group 3 (copper sulfate with Azolla) and group 4 (copper sulfate with water Duckweed plant) (1.098±0.0816, 1.020±0.164), respectively. while group 5 (copper sulfate with Azolla and Duckweed combo) 0.922±0.166 compared to group 1 (controls) 0.944±0.101. In addition, total protein levels were significantly higher in group 2 (copper sulfate) 33.4±1.023, followed by group 4 (copper sulfate with water Duckweed plant), group 5 (copper sulfate with a mixture of Azolla and Duckweed), and group 3 (copper sulfate with Azolla) 29.004±3.235, 28.86 0.697, and 24.598±1.027, respectively, compared to controls. The levels of albumin were found to be greater in group 2 (copper sulfate). As well as lower in the fifth group (copper sulfate with a combination of Azolla and Duckweed), with 10.564±0.548, 10.134±0.664, and 9.95±0.169, accordingly, compared to group 1 (controls) at 11.334±0.677. Furthermore, the results revealed significantly higher of albumin, the results reveled that higher level of albumin in group 2 (copper sulfate) 21.296±0.846, followed by group 4 (copper sulfate with water Duckweed plant), group 5(copper sulfate with a mixture of Azolla and Duckweed) and group 3(copper with Azolla) 18.902±3.083, 18.29±1.090, 14.464±0.78 respectively compared to group 1 (controls) 11.334±0.928 (Table 3)

Table 3: Triglyceride, Total protein, Albumin, Total cholesterol, and Globulin levels among study groups

Total Cholesterol (mmol/L), Triglyceride (mmol/L). Total protein (gm/L). Albumin (gm/L). Globulin (gm/L). *Data as Mean ± slandered errors. P<0.05.

Histopathological result

The following conclusions were drawn from the examination of histological sections and the histological damage score. Histopathological examinations revealed that fish that had been repeatedly exposed to various extracts had severe structural abnormalities in their gills and intestine. The gills of a fish from the CuSO₄ treatment group in this study were investigated histologically. This demonstrated that C. carpio gill subjected to CuSO₄ for 56 days exhibits hyperplasia of mucus cells with edema and sloughing of epithelial cells (Figure 3). A microscopic study of the gills of C. carpio subjected to CuSO₄ reveals hemorrhage as well as hydropic degeneration of epithelial cells (Figure 4). A microscopic analysis of the gill in C. carpio subjected to CuSO₄ and Duckweed reveals normal interlamellar space (Figure 5). On the other hand, histological examination of C. carpio kidney subjected to CuSO₄, Azolla, and Duckweed revealed loss of straight secondary gill filaments as well as hydropic degeneration of epithelial cells (Figure 6).

A histological examination of the gut of a CuSO₄ treated fish reveals severe hydropic deterioration of epithelial cells lining the villi, as well as damage and sloughing of the villi apex (Figure 7). Furthermore, a microscopic examination of the gut of C. carpio subjected to CuSO₄ reveals infiltration of inflammatory cells with adhesion between villi, as well as extensive hydropic deterioration of epithelial cells (Figure 8). Furthermore, the intestine of C. carpio subjected to CuSO₄ and Duckweed water plant shows edema in the submuscularis layer (black arrow) with minor adhesion between villi (Figure 9). The microscopic examination of the group treated with CuSO₄ and Azolla reveals enteritis with mucus secretion (Figure 10). The water plant in the group subjected to CuSO₄ and a mixture of Azolla and Duckweed showed mid enteritis involves hydropic degeneration in the gland's epithelial cells (Figure 11).

Figure 3: The gills of C. carpio subjected to CuSO₄ for 56 days display hyperplasia of mucus cells (black arrow) with edema (red arrow). sloughing of epithelial cells (green arrow), H&E, 40×9.1X.

Figure 4: microscopic examination of gills in C. carpio exposed to CuSO₄ for 56-day show hemorrahge (black row) with hydropic degeneration of epithelial cells (red row). H&E, 10×5.1X.

Figure 5: microscopic examination of gills in C. carpio exposed to CuSO₄ with Duckweed for 56 days show normal interlamellar space (black arrow) H&E, 10×7.5X.

Figure 6: microscopic examination of gills in C. carpio exposed to CuSO₄ with Azolla and Duckweed for 56-day show loss straight secondary gill filaments (black arrow) with hydropic degeneration of epithelial cells (black dot arrow), H&E, 40 ×4.8X.

Figure 7: A microscopic analysis of the gut of C. carpio subjected to CuSO₄ for 56 days reveals severe hydropic degeneration. epithelial cells lining villi (black arrow) with damage and sloughing of the villi, apex (red arrow). H&E, 40×1.8X.

Figure 8: microscopic Photograph of intestine in C. carpio exposed to CuSO₄ for 56 days show inflammatory cell infiltration (black arrow) with villi attachment (red two head arrow), severe hydropic degeneration in epithelial cells (yellow arrow) H&E,40×3.5X.

Figure 9: Photograph of intestine in C. carpio exposed to CuSO₄ with Duckweed for 56-day show edema in the submuscularis layer (black arrow) with mild adhesion between villi (red arrow) H&E, 10×5.1X.

Figure 10: Photograph of intestine in C. carpio exposed to CuSO₄ with Azolla for 56 days show enteritis (black arrow) with mucus secretion (red arrow) H&E, 10×3.1X.

Figure 11: Photograph of intestine in C. carpio exposed to CuSO₄ with Azolla and Duckweed for 56 days show mid enteritis (black arrow) with hydropic-degeneration in the epithelial cells lining gland (red arrow) H&E,40 ×4.3X.

Discussion

Fat is a crucial nutrient providing energy for life activities, and its fatty acid profile is critical in human and living organisms, as it can either protect against or induce coronary heart disease (28-30). Tang et al. (31) displayed that palmitic acid, linoleic acid and linolenic acid were the dominant components of fatty acid in Duckweed. Mary et al. (18), reported for fatty acids profile Duckweed, the PUFA and MUFA were significantly higher comparted to the saturated fatty acids. Azolla contains significant levels of fatty acids, including MUFA, PUFA and SF, this lipid fraction can be used to make high quality biodiesel, but this requires an additional fractionation stage and for the growth and proper performance of fish (32).

 Recent research in nutrition and food science has emphasized the potential antioxidant, antimicrobial, and anti-inflammatory activities of plant products, including flavonoids and phenolic compounds, which are crucial natural compounds that can prevent coronary heart disease (33). A study by Ilhami et al. (34) reported that Duckweed found to be effective antioxidants due to high content of phenolic and flavonoids. Al-Khafaji et al. (29) pointed that Azolla includes several important phytochemicals such as flavonoids and phenols, these bioactive compounds contribute to therapeutic properties including anti-inflammatory, antioxidant, anti-hypertensive and antidiabetic, the presence of hydroxyl group in these compounds effective scavenging of free radicals. Many manufactured and natural chemicals dissolve in freshwater. Fish can be exposed to a wide range of pollutants. such as heavy metal salts such as CuSO₄, and have evolved to the effects of naturally occurring compounds in the water. Copper can accumulate in fish organs, causing redox reactions that produce free radicals, disrupting various physiological processes and altering morphology (35). Copper sulphate, a chemical copper, is utilized in industrial and agricultural applications. processes as a component of pesticide formulations. Numerous studies have demonstrated the effects of this chemical at varying dosages, including behavioral abnormalities,

decreased development, and reproductive issues (36,37). These findings are consistent with a study in which researchers concluded that copper sulfate might increase total cholesterol levels in mice (38,39). One study reported that tilapia fish in the experimental group (which received Azolla) had lower levels of total cholesterol compared to tilapia fish in the control group. This may be because Azolla contains compounds called sterols, which are similar in structure to cholesterol. Sterols can reduce the absorption of cholesterol from the intestine. In addition, Azolla is rich in fiber, which can help lower cholesterol levels by binding cholesterol in the intestines and removing it from the body (40,41). Copper may also cause physiological changes and protein dysfunction. Excessive copper exposure in fish can damage the gills, intestines, and sensory system (42,43). Oslo Azolla concede as Metal- cleansed water. It was revealed that metals (iron and copper) can be removed by A. pinnata and L. minor (Duckweed) (44,45). They concluded that mildly polluted wastewater may be cleaned by running it through ponds holding one or both of these water plants. Saxena discovered that a 2:1 blend of Lemna and Azolla may adequately filter substantially contaminated manufacturing wastewater for agricultural application (46,47).

Conclusion

 The study substantiated the nutritional value of Duckweed and Azolla meal in the diets of C. carpio communis young. It appears to be an acceptable substitute for fishmeal in world diets because it has no negative effects on the histopathology or blood biochemical markers of C. carpio communis. With its intensification, Azolla and Duckweed meal might be successfully introduced to provide inexpensive and environmentally acceptable diets for raising large quantities of C. carpio communis fingerlings.

Conflict of interest

None.

Acknowledgments

The thanks extended to College of Agriculture and Forestry, University of Mosul, to support current study.