Evaluation of Different Quantity of Iron Oxide Nanoparticles on Growth, Haematological and Biochemical Characteristics of Koi Carp

Aquaculture is the world’s fastest-growing sector of agriculture. Presently, India ranks second in terms of total fish production with an annual fish production of about 9.06 million metric tonnes. Among aquaculture, ornamental fish culture is the culture of attractive colorful fishes. Among different ornamental fishes, Koi carp is one of the most popular, favorite ornamental fish and it has high market value because of its excellent color. The fisheries and aquaculture industry can be revolutionized by using nanotechnology tools for rapid disease detection, targeted delivery of drugs, DNA vaccines and nutrients. Nanoparticles such as Se, Cu, Fe, FeO, Zn and ZnO play a vital role in an aquaculture operation. The dietary supplementation of nanoparticles produced better survival, growth, hematology, biochemical, antioxidant levels and immunity in aquatic organisms. Iron is necessary by most of the living organisms because it is required for the metabolic process including oxygen transport, drug metabolism, steroid synthesis, DNA synthesis, ATP production, electron transport and cellular respiration (Crichton et al., 1991). The iron content of fish is very low compared to that of mammal. Although the gill membrane absorbs iron to a certain extent, the intestinal mucosa is considered to be the major site. The work related to the evaluation of different quantities of iron oxide nanoparticles on growth, hematological and biochemical characteristics of Koi carp is wanting. Hence the present work was carried out.

Synthesis and characterization of iron oxide nanoparticles
 
The co-precipitation method was used for the synthesis of iron oxide nanoparticles. The aqueous solution of FeCl₂ and FeCl₃ was prepared in a 1:2 ratio. NaOH (0.1N) was added drop by drop under constant stirring for 30 minutes, the solution turns brownish yellow color. The yellow color of the aqueous solution turned into greenish-black precipitated by the addition of NaOH when the pH was 12. Then it was centrifuged at 5000 rpm for 3 minutes with ethanol and the collected iron oxide nanoparticles were allowed to air dry/dissolve with distilled water for 8 hr sonication (50Hz frequency Vibronics -230V) (Fig 1). The morphology of the Fe2O₃NPs was determined by using a scanning electron microscope (SEM) (LEO 1455 VP). An energy-dispersive X-ray detection instrument (EDAX) (HORIBA 8121-H) was used to examine the elemental composition of the sample. Structure and crystalline size of Fe₃O₄ nanoparticles were determined by XRD using an X-ray diffractometer with nickel –filter CuKa radiations in the 20 range (l=1.5418Å) from an X-ray tube run at 40kV and 30ma. The FT-IR measurement was carried out to identify the functional groups of the bioactive components based on the peak value in the region of infrared radiation. The magnetic property of Fe₂O₃ nanoparticles was determined by a vibrating sample magnetometer (VSM).
 

 
Collection and acclimation of Koi carp
 
For growth work, Koi carp fish fingerlings (1.504 ± 0.30g) were collected from Aqua garden, Kadachanenthal, Madurai, Tamil Nadu, India and transported to the laboratory in polythene bags filled with oxygenated water. Fishes were acclimated in glass aquaria (60×45×45 cm) for 15 days at 28±2°C and fed with trainee feed containing fish meal, groundnut oil cake, wheat flour and rice bran in the form of dry pellets.
 
Selection of feed ingredients and experimental feed preparation
 
The raw materials are selected based on their ability to supply nutrients such as proteins, carbohydrates and fat at the low cast. Fish meal and Groundnut Oil Cake were used as protein sources; wheat flour and tapioca flour were used as carbohydrate sources; vegetable oil used as lipid source and served as binding agents and suppletive mix were used. After knowing the protein content by the Micro-Kjeldahl method (Jeyaraman, 1992) the raw materials were ground and sieved through a 425-micron sieve. The ingredients were weighed and mixed thoroughly with 130-150ml of distilled water. The mixed feedstuff was put in an autoclave for 15min at 100°C and cooled. After cooling, fish oil, sunflower oil, suppletive-mix, sodium chloride, sodium benzoate and different quantity of iron oxide nanoparticles such as 0, 10, 20, 30, 40 and 50mg/100g^-1 were mixed for feed I, II, III, IV, V and VI and it was extruded with the help of pelletizer. The pellets were dried at room temperature. The formulated feed was kept in an airtight container at -20°C until used to prevent contamination (Table 1).
 

 
Design for growth studies
 
 For the present work, the uniform size of Koi carp (Cyprinus carpio var.koi) (1.504±0.30 g) were selected and introduced in rectangular glass tanks (45cm×22cm×22cm) having a capacity of 18 liters. Five fishes were introduced in each tank and each treatment triplicates were maintained. During rearing, the fishes were fed on the ad-libitum diet of the prepared feed twice a day for 1 hour each from 9-10 am and 4-5 pm. The unfed were collected after one hour of feeding without disturbing the fishes and dried to constant weight. The fecal matter was collected daily before changing the water with the least disturbance to the fishes and dried at 95°C. Approximately 70% of the water in the tank was replaced with tap water. The experiment was continued for 21 days. On the 21^st day, the length and weight of the fishes were measured in live conditions. Collected Muscle, Gill and Liver from all treatments for further analysis. Condition factor (k), survival rate and feed utilization parameters such as feed consumption, feed conversion efficiency, feed conversion ratio, specific growth rate, assimilation, metabolism, gross growth efficiency and net growth efficiency were estimated after 21 days.
 
Hematological assay
 
Blood samples were collected from the anus point on the right side of the fish. Complete blood parameters such as WBC, Hemoglobin, RBC, Hematocrit (PCV), MCV, MCH, MCHC and platelet count were estimated.
 
Biochemical characteristics of muscle, gill and liver of Koi carp
 
Total protein (Lowry et al., 1951), carbohydrate (Carrol et al., 1956) and lipid (Barnes and Blackstock, 1973) were estimated.
       
The whole work was carried out in the Department of Biology, The Gandhigram Rural Institute- Deemed to be University, Gandhigram, India between September 2018 and April 2019.

Iron oxide nanoparticles were synthesized by co-precipitation of Fe2+ and Fe3+ by the addition of a base. The size, shape and composition of iron oxide nanoparticles synthesized through chemical methods depend on the type of salt used, Fe2+ and Fe3+ ratio, pH and ionic strength (Ali et al., 2016). Complete precipitation of Fe₃O₄ was expected between pH 9 and 14 (Gupta and Wells, 2004) and the precipitate was black (Cornell and Schertmann, 1991). Scanning Electron Microscopy indicates that nanoparticles formed as agglomerated because of the adhesive nature of distorted irregular cluster appearance (Fig 2). The diameter of iron oxide NPs is calculated in the range of 50-90 nm where the average diameter of iron oxide NPs is close to 60+10 nm. Poedji Loekitowati Harini et al., (2013) reported that the SEM image of synthesized iron oxide nanoparticles has a clear image of the cluster shape ranges from 30nm to 100nm. EDAX spectrum recorded on the iron oxide nanoparticles is shown as two peaks located between 0.5 KeV and 8 KeV (Fig 3). The maximum peak located on the spectrum at 6.4 KeV clearly shows iron. The second maximum peak located on the spectrum at 0.5 KeV indicates oxygen. The composition of iron and oxygen is 70.86% and 29.14%. Keerthika et al., (2017) reported that the EDAX spectrum iron oxide nanoparticles show three peaks located between 2KeV and 10KeV. The phase structure of synthesized iron oxide nanoparticles is shown in Fig 4. The 2θ value of 24.150, 33.150, 35.610, 40.790, 54.070 and 64.060 in the reference element iron (JCPDF No: 089-8104) which are attributed 012, 104, 110, 113, 024, 116, 214 and 300 crystallographic plane of face-centered cubic iron crystals. These results indicated that iron oxide nanoparticles are cubic and also supported by the XRD results which indicate that the γ-Fe₂O₃ phase contains only Fe3+ cations. Furthermore, the approximate crystallite size (D) of the iron oxide nanoparticles was calculated using the Scherer equation and approximate particle size 20+25 nm. Carmen et al., (2012) also reported γ-Fe₂O₃ and the particle size was calculated using the Scherer equation. FT-IR measurement was carried out to identify the functional groups of the bioactive components based on the peak value in the region of infrared radiation. Iron oxide formation was confirmed by 3456.24, 2928.47, 1628.25 and 555.13 cm^-1 bands have -OH stretching, primary amines, alcohol, phenols and alkyl halides. (Fig 5). Arokiyaraj et al., (2013) reported that the main functional group of iron oxide nanoparticles are alcohol, phenols and primary amines.  The magnetic properties of synthesized nanoparticles in the presence of a magnetic field were measured using a vibrating sample magnetometer. Fig 6 shows that the saturation magnetization (Ms) of Fe₂O₃ nanoparticle is 1.019 emu/gm. Ying Wang et al., (2013) also reported that the saturation magnetization (MS) is 1.019 emu/gm for iron oxide nanoparticles. Ferromagnetic properties were already reported by many authors in different methods (Pei et al., 2007, Lopez et al., 2012 and 2014 and Zhuang et al., 2014).
 

 

 

 

 

 
The condition factor was higher in Feed III when compared to other iron oxide nanoparticles supplemented feeds (Table 2). Srinivasan et al., (2016) reported an increase in the condition factor of Macrobrachium rosenbergii post-larvae fed with 40g/ kg^-1 of iron oxide nanoparticles in the feed. Survival, feed utilization and growth parameters of Koi carp about the different quantity of Iron oxide nanoparticles are presented in Table 3. Survival rate of Koi carp was 80 to 90% in 20, 30 and 50mg of iron oxide supplemented feeds. A similar survival rate was also reported in common carp fed with iron oxide nanoparticles (Anand Sadanandan Ramya et al., 2014). Feed consumption and feed conversion efficiency of Koi carp were higher in feed VI (3.26 + 0.57and 0.28 +0.08) containing 50 mg /g^-1 of iron oxide nanoparticles and lower in feed I (control). Amina Zuberi et al., (2015) reported that the feed consumption and feed conversion in Juvenile Grass Carp (Ctenopharyngodon idella) increased with an increase in the concentration of Zinc nanoparticles. Hayat et al., (2007) also reported the increase of growth parameters except feed conversion ratio in major carps viz. Catla catla, Labeo rohita and Cirrhinus mrigala. The feed conversion ratio was good in Ex. Feed IV (3.15) containing 30 mg of iron oxide nanoparticles. Mukesh Mehta Ambani (2015) reported that the feed conversion ratio was higher in control when compared to different concentrations of prepared feed of Macrobrachium rosenbergii. Muralisankar et al., (2016) reported that the feed conversion ratio was higher in control and lower zinc oxide fed with Macrobrachium rosenbergii. Growth and Specific Growth rate were higher in Feed IV (30 mg/g^-1) when compared to control and significantly increased the Koi carp growth. Davis et al., (1993) and Gammanpila et al., (2007) also reported that the specific growth rate was gradually increased in lower concentration to a higher concentration of zinc supplemented feed of Penaeus vannamai. The assimilation of Koi carp is higher in feed IV and metabolism is higher in feed II. Gross growth efficiency of Koi carp is higher in feed IV and net growth efficiency of Koi carp is higher in feed V. Feed consumption, growth, gross and net growth efficiency of Koi carp is significantly varied (Table 4).
 

 

 

       
Hematological parameters are very helpful in the judgment of the health condition of fish species. The complete blood count of Koi carp progressively increased when the quantity of iron oxide nanoparticles increased (Table 5). Anand Sadanandan Ramya et al., (2015) reported that the hematological parameters were gradually increased with different doses of iron oxide nanoparticles fed on Indian major carp. Abdel et al., (2007) also reported the increase of blood parameters when compared to control with a high concentration of selenium nanoparticles supplemented feed to African catfish, Clarias gariepinus. Saravanan et al., (2015) reported that the hematological parameters of Labeo rohita exposed to 1 and 25 mg/l of Fe₃O₂ Nps showed a significant (P<0.05) decrease.
 

       
Total protein, carbohydrate and lipid of muscle, gill and liver of Koi carp gradually increased when the concentration of iron oxide nanoparticles increased (Table 6). Keerthika et al., (2017) reported that the iron oxide nanoparticles altered the biochemical parameters of Labeo rohita. Muralisankar et al., (2016) reported the concentration based increase and decrease of protein, lipid and carbohydrate contents in Zn supplemented diet fed to Macrobrachium rosenbergii. Ashouri et al., (2015) also reported that the selenium nanoparticles in the feed have increased the protein, carbohydrate and lipid content of muscle, gill and liver of Crucian carp (Carassius auratus).
 

The findings strongly suggest that feed IV containing 30mg Iron oxide nanoparticles was sufficient for the growth of Koi carp and feed VI containing 50 mg of Iron oxide nanoparticles are suitable for the enhancement of hematological and biochemical parameters.

The authors declare no conflict of interest.

Authors thank the Department of Biology, The Gandhigram Rural Institute-Deemed to be University, Gandhigram, Tamil Nadu, India for offering laboratory facilities to carry out this work.