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Effects of calcium carbonate nanoparticles on water quality, growth and metabolic activity of Macrobrachium nipponense in zero-water exchange biofloc system

The purpose of the present research was to investigate the effects of adding calcium carbonate nanoparticles to the Macrobrachium nipponense diet in the biofloc system under zero exchange conditions. Oriental River prawn (inital weigh of 0.82  ±  0.07 g) were divided into four groups and fed four levels of calcium carbonate nanoparticles as following 0, 25, 50 and 100   mg kg diet in biofloc system (CN0, CN25, CN50, and CN100) for 28 days. This study was applied complete randomized design with three replications. Water quality parameters were measured during the test period. Feed and growth parameters and some metabolic activities of hepatopancreas were measured. Physico-chemical water factors were in the appropriate range for this species. The concentrations of total ammonia nitrogen (TAN), nitrite, and nitrate were not significantly different between the experimental groups. Correspondence H. Adineh, Department of Fisheries, Faculty of Agriculture science and natural resources, Gonbad Kavous University, Gonbad, Iran (e-mail: adineh.h@gonbad.ac.ir). The growth of prawns was significantly higher and feed conversion ratio was lower in CN25 and CN50 groups compared to the control group. The lowest AST and ALT activities were observed in CN25 and CN50 groups compared to the control. The prawns fed with experiment diets had significantly higher total protein, hemocyanin, glucose, and calcium compared to the control. Overall, the results showed diets containing Nano-calcium carbonate at levels 25-50 mg kg in CN25 and CN50 groups could improve growth performance and metabolic activity of oriental river prawn in the biofloc system.


Introduction
Freshwater prawn farming plays an important role in the global aquaculture industry.
Oriental river Macrobrachium nipponense is widely distributed in freshwater and lowsalinity areas of rivers. Oriental river M. Biofloc technology (BFT) is a suitable and useful system based on the growth of microorganisms. Microorganisms play two major roles in maintaining water quality (by absorbing nitrogen compounds in the production of microbial protein) and nutrition (increasing production per unit area and reducing feed conversion ratio) in the aquaculture environment (De Schryver et al., 2008;Avnimelech, 2009;Crab et al., 2012).
The main advantages of the culture system with biofloc technology are the use of nitrogenous wastes, the limited use of water and at least wastewater is released to the environment (Avnimelech, 2009;Emerenciano et al., 2011), and so BFT is environmentally friendly. In addition, BFT systems enhance the growth parameters, feed performance, enzymatic activities, and immune and also antioxidant indexes of cultured shrimp (Xu et al., 2013;Kumar et al., 2017;Panigrahi et al., 2020). In this regards, research has been reported on the effects of BFT on the growth and liver histology changes of Speckled shrimp (Metapenaeus monoceros) (Kaya et al., 2019) and Pacific white shrimp Litopenaeus vannamei (Xu and Pan, 2012;Khanjani et al., 2017), the effects of different types of feeds and salinity levels on Pacific white shrimp juveniles in a biofloc system (Khanjani et al., 2020), and the effects of different feeding levels on Pacific white shrimp in zero water exchange system (Khanjani et al., 2015).
Divalent cations such as calcium (Ca 2+ ) are responsible for the formation of bioflocs by improvements in floc properties as measured by sludge volume index (SVI), capillary suction time (CST), specific resistance to filtration (SRF), cake solids, and floc strength (Sobeck and Higgins, 2002). Calcium is an essential element in the body that is required for bone formation, growth, cellular physiology, immune response, and blood coagulation (Reid et al., 1993). Calcium carbonate is the most common form of calcium supplements (NRC, 1994 Therefore, this project was planned to improve the technical production of BFT and to provide a more sustainable system for shrimp culture.
In our study, the effects of feeding M.
nipponense with different levels of calcium carbonate nanoparticles in the biofloc environment were evaluated.

Prawns
Oriental river Macrobrachium nipponense were caught with a hand-held saucer around coastal waters of Golestan Dam located in Gonbad Kavous, Golestan Province, Iran. All prawn samples were immediately transferred to the aquaculture laboratory at Gonbad Kavous University. Prawns were acclimatized with laboratory conditions for 7 days. The prawns (inital weigh of 0.82 ± 0.07 g) were distributed into 12 aquaria (volume of 25 L) and reared for 28 days. The photoperiod was a 10 h:14 h light-dark cycle.

Biofloc Production
Biofloc production was performed in two fiberglass tanks with a water volume of 40 liters in the laboratory environment. In each tank, three air stones were created to make a circular flow of water for complete mixing.
Biofloc material contained commercial feed, flour wheat, urea, and molasses. Previously produced microbial flocs were inoculated in the biofloc system at a rate of 10% of the total volume (Martins et al., 2017). The carbon: nitrogen (C:N) ratio was maintained at 15:1 using the carbon source (molasses) described by Crab et al. (2012). According to Craig and Helfrich (2002), 16% of protein is N. If the prawns are consumed 100 g of feed with 34.30% protein, the N amount consumed by the prawns is 5.48 g (Asaduzzaman et al., 2008) and 4.11 g N ends up in water. On average 75% of the feed-N ends up in the water (Piedrahita, 2003). The C:N ratio was maintained at 15:1, so 61.65 g C per 100 g of feed needed for biofloc production. The amount of carbon source added will then depend on the C content of the carbon source.
In case of molasses (containing 0.4 g C per g), 154.12 g of carbon source would be needed (Sierra-De La Rosa, 2009).

Experimental diets
The diet ingredients were prepared and uniformly mixed (

Metabolic activity
Feeding was stopped 12 hours before sampling. All prawns were counted and weighed after collection. In each tank, hepatopancreas from ten prwan were separated to measure metabolic activity. The total protein, glucose, calcium, cholestrol, AST, ALT, and hemocyanin were determined using commercially available kits. Total protein was determined based on Shi et al. (2006).
According to the method of Kunst et al.

Statistical
Data were presented as mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple range tests.
Before analysis, normality and homogeneity of variance were checked with the Shapiro-Wilk and Levene's tests, respectively. The differences were measured in statistical with SPSS at p < 0.05 level.

Results
The results of water quality parameters in the experimental groups during 28 days trial are shown in Table 2. Phosphate in CN25 group was higher than CN50 and CN100 groups. Also, BFV in CN100 group was higher than CN25 group. The water variables ranges were into the acceptable levels for shrimp culture. Data are presented as mean ± SD. Data were analyzed through one-way ANOVA besides Duncan comparisons. Data with different superscript letters (a, b and c) in the same row mean significant differences among experimental groups (p < 0.05).
The fish growth performance and feed efficiency are presented in    Data are presented as mean ± SD. Data were analyzed through one-way ANOVA besides Duncan comparisons. Data with different superscript letters (a, b and c) in the same row mean significant differences among experimental groups (p < 0.05).

Discussion
The environment and farming system are main factors that affect the growth performance and If the ratio of carbon and nitrogen is regulated and balanced in the culture environment, nitrogen compounds are converted into bacterial biomass (Schneider et al., 2005). In our study, water parameters were appropriate for Macrobrachium rearing (Ballester et al., 2017). Water factors such as temperature, pH, and dissolved oxygen showed that no   (1)