QUALITY OF CARP MEAT ( Cyprinus carpio ) PRODUCED IN A POND WITH THE ADDITION OF PURIFIED WASTEWATER ORIGINATING FROM THE SLAUGHTERHOUSE

Th e aim of the study was to investigate production parameters and nutritive quality of carp meat produced in a fi sh pond fed with fresh well water mixed with purifi ed slaughterhouse wastewater. Th e quality of carp meat was analysed in view of seasonal variations in chemical composition of fi llets. Our research idea was that purifi ed slaughterhouse wastewater would provide essential nutrients for carp and positively aff ect the quality of fi sh meat, which was confi rmed in this experiment. Th e total carp production at the end of study period was 3,270 kg/ha. Th e research revealed a feed conversion ratio (FCR) of 1.5 kg of feed per one kg weight gain and a survival rate of 87%. Statistically signifi cant diff erence ( p < 0.05) between meat protein content during spring and autumn season was established. Protein content in meat was higher during spring season, whereas higher fat content was established during autumn sampling season. Th e application of purifi ed slaughterhouse wastewater provided high level of nutrients resulting in high meat yield per area unit and good chemical composition of meat. Such production model is meaningful in both economic and ecological aspect.


INTRODUCTION
In some regions, wastewater from slaughterhouses is still released into the rivers, channels, lakes and other water bodies without adequate pre-treatment. Th is causes substantial hazard for the environment. Such wastewaters contain high quantity of organic matter that represents an excellent source of nutrients for carp species. Vo (2001), Th i Phong Lan et al. (2007) and Pelić (2020) reported that such wastewaters can be used in aquaculture as a source of both water and important nutrients for fi sh. Th e use of purifi ed wastewater in fi shponds is an innovative recycling method implicating conversion of soluble nutrients into biomass, that is, fi sh. It can be considered a completely novel approach to providing sustainability of meat industry and environment protection (Pelić, 2020). Carp is an omnivorous fi sh species that feeds on natural food available in the pond, which makes it particularly suitable for farming in such systems. Fish meat is a valuable source of nutrients, which is essential for variable and healthy human diet. An optimal ratio of proteins, fats, carbohydrates, minerals and vitamins contributes to a high nutritive value of fi sh meat (Ćirković et al., 2011). It contains up to 85% water, 16 to 22% proteins, 1 to 20% fat and 0.8 to 2% minerals (Ljubojević et al., 2013a;2013b). Th e amounts of nutrients in fi sh meat are highly variable depending on a wide range of factors such as genetic, species, age, diet, gender, health condition, season of the year, water quality, farming system, etc. (Marković et al., 2016;Ljubojević Pelić et al., 2018). Proteins from fi sh are characterized by good amino-acid composition with substantial amounts of free amino acids (Buchtová et al., 2010). Fish proteins contain all aminoacids essential for human body and can be used as the sole source of protein in the diet (Vladau et al., 2008). Fish meat contains much less connective tissue comparative to the meat of endothermic animals, which contributes to its better digestibility preferred by specifi c consumer categories (Vladau et al., 2008). Fish contains negligible amounts of carbohydrates in the form of glycogen and high water content (60-86%) (Ćirković et al., 2011). Fish meat fat content is highly variable. Accordingly, based on their fat content, fi shes are categorized into lean (< 5% fat), semi-fat (5 -10% fat) and fat (> 10% fat) (Ljubojević et al., 2013b). Being a most valuable source of polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that can only be eff ectively synthesized by aquatic organisms, consumption of fi sh meat is the only way to intake the aforementioned fatty acids by humans (Pal et al., 2018). Th us, regular use of fi sh in the diet is highly recommended.
Fish consumption is benefi cial and associated with lower risk of dementia and Alzheimer disease, and protective eff ects against some cancers are suggested (Connor and Connor, 2010). Regular intake of fi sh decreases the risk from cardiovascular diseases, especially myocardial infarction, atherosclerosis, hypertension and other (Kris-Etherton et al., 2002). Positive eff ects of fi sh in human diet are established in view of prenatal development, preservation of functions of nervous system, eyes and skin (Allen and Harris, 2001). Specifi c fl avor, taste and good digestibility contribute to overall acceptability of carp meat.
Considering the aforementioned, investigating the options of fi sh farming by using purifi ed wastewater is highly supported. Th e lack of available literature data on production parameters and meat quality of fi sh reared in ponds fi lled with purifi ed wastewater encouraged our research towards determining production parameters and meat quality of carps farmed under the described conditions. To that end, carp fi llets were analysed for basic quality parameters. Moreover, the carp fi llet samples were examined during two sampling periods in order to identify potential seasonal eff ects on meat quality.

MATERIAL AND METHODS
A 1 ha fi sh pond with average depth of 1.3 m was set up as a part of the meat industry "Đurđević" in Pećinci providing an integrated production structure including slaughterhouse, wastewater purifi cation system and fi sh pond. Th e process of water purifi cation was performed in a wastewater treatment system. Th e purifi cation procedure includes physical, biological and chemical processes. First, untreated wastewater was mechanically treated. Aft erwards, water fl ows into the aeration tank and undergoes biological processing. Finally, the wastewater is discharged into the secondary wastewater treatment tank and chemically treated. Th e pond was built at the high-quality arable land and in line with basic principles and requirements described by Ćirković et al. (2002). Th e pond is supplied with both well water and purifi ed slaughterhouse wastewater under continuous aeration. Th e ratio of water from the well and purifi ed water from the slaughterhouse was about 2:1. In the fi rst year, two-year old carps purchased from commercial ponds were introduced with a stocking density of 2,500 individuals/ha. Th e average initial weight of carps was 200 g. Fish were fed mixture of grains and edible off al (spleen, liver) of slaughtered animals supplemented with complete carp-feed mixtures. Edible off al was heat-treated (cooking) at a temperature of 90 °C. Th e off al has been fi nely ground and mixed in a cutter with the addition of cereals until obtaining a doughy consistency. Th e relationship between fresh feed (grains and edible off al) and complete mixtures in spring was 1:1. Th e ratio in fresh feed used was 30% for grains and 70% for edible off al. At the end of the fi rst year, the survival rate was 74%. Production parameters were calculated according to fi sh biomass and feed conversion ratio described Abdelghany and Ahmad (2002). Th e fi rst sampling was carried out in April next year, and the second one in the same year in October, that is, at the end of experimental period. In the second year, the survival rate was 87%. During the last three months, only grains and complete mixtures were added in the ratio 1:1. Th e samples of three-year-old common carp were transported to the laboratory in a transport cooler. All samples were fi lleted before analysis.
Chemical composition of fi sh was examined applying standard SRPS ISO methods. Gravimetric method was utilized to determine the moisture content (SRPS ISO 1442:1998) and total fat (SRPS ISO 1443:1992); total protein content was determined combustion method (AOAC Offi cial Method 992.15). Ash content was determined by combustion at 550±25 ºC applying standard method SRPS ISO 936:1999. Salt concentration was determined applying standard method SRPS ISO 1841-1:1999. Hydroxyproline level was measured spectrophotometrically applying SRPS ISO 3496:2002. Subsequently, collagen content and collagen content in total protein were calculated. Energy value was expressed per 100 g of carp fi llet, and was calculated according to the equation below using conversion factors indicated in the Appendix 13 of the Rulebook on declaration, labelling and advertising of food ("Offi cial Gazette of RS", No. 19/2017, 16/2018, 17/2020 and 118/2020): Energy value (kcal/g) = 4 × carbohydrate content + 4 × protein content + 9 × fat content.

Statistical data analysis
Th e data were analysed using Excel (Microsoft Excel 2007, Microsoft Corporation, Redmond WA, USA) soft ware package and Data Analysis. Student's t-test was applied to compare the arithmetic mean values of the results obtained for samples collected in spring and autumn. Th e data were expressed as mean values ± SD. A p-value of p < 0.05 was considered a statistically significant diff erence. Besides, Pearson correlation coeffi cient was used as well.

RESULTS
Production parameters were determined during harvesting at the end of study period. Total carp production per area unit was 3,270 kg/ha. Feed conversion calculated as the amount of supplementary feed spent for 1 kg weight gain was around 1.5 kg. Survival rate was high, reaching 87%. At spring sampling, in April in the second year of the study, the carp body weight ranged from 695 to 1,675 g (average weight 820 g), whereas values at autumn sampling in October in the second year of the study ranged between 1,710 and 2,700 g. At the end of experimental period in October in the second year of the study, the average live body weight of three-year old common carp was 2,020 ± 252.3 g.
Chemical composition of carp fi llet is shown in Table 1. Higher protein content (17.99 g/100g) in fi llets was determined in spring period. Th e diff er-ence between spring and autumn protein content was statistically signifi cant (p < 0.05). Th e obtained results revealed higher fat content in the fi llets during autumn period (5.19 g/100g) as compared with the spring season; however, the diff erence is not statistically signifi cant (p > 0.05). Salt content was significantly higher at spring sampling (p < 0.05). Th e diff erences in energy value of the fi llets analyzed in spring and autumn period were not statistically significant (p > 0.05). Th e results were expressed as mean values ± SD (n = 7); p < 0.05

DISCUSSION
To our knowledge and according to the available literature the data on production performance and meat quality of carps reared in purifi ed wastewater are pretty sparse, which suggests the innovative character of our study. Th e obtained results on production parameters correspond with the production parameters of carps sampled in conventional fi shponds in the Republic of Serbia. Total carp production per area unit recorded in this study was 3,270 kg/ha. In fi shponds with semi-intensive production system, where fi sh is fed naturally available feed resources supplemented with grains, the production performance is up to 1,500 kg/ha (Marković et al., 2016). Rearing fi sh in good production conditions and the use of high-quality concentrated extruded feed would off er possibility of increasing production performance and yields that may reach over 3,000 kg/ha (Stanković et al., 2011).
Adequate knowledge about fi sh meat quality is of importance in view of its role and value in human diet (Kris-Etherton et al., 2002, Connor and Connor, 2010). Our research revealed that moisture content in carp meat samples collected in spring season (74.95 ± 1.37%) was signifi cantly lower (statistical signifi cance p = 0.03) as compared to values measured in autumn period (76.16 ± 1.27%). Protein content determined in spring period (17.99 ± 0.40%) was higher than that observed in autumn (17.67 ± 0.29%), which is considered statistically signifi cant (p = 0.03). Fat content in spring (4.57 ± 1.35%) was lower than that in autumn season (5.19 ± 1.55%); however, the diff erence was not statistically signifi cant (p = 0.29). Higher contents of fat in autumn period could be attributed to the increased body-size of the fi sh. Also, nutrition as well as the types of fi sh feed play a very important role in the fat content in the body of fi sh (Stanković et al., 2011;Trbović et al., 2013;Marković et al., 2016). Some earlier researches have proved high correlation between the body-size of the carp and fat content in the meat (Kocour et al., 2007). A negative correlation was observed between fat and moisture contents in carps. Pearson correlation coeffi cients for the aforementioned parameters were 0.97 and 0.96 in spring and autumn season, respectively, which indicates a high negative correlation. Collagen contents determined in spring and autumn were 1.04 ± 0.43% and 1.07 ± 0.13%, respectively, with no statistically signifi cant diff erences (p = 0.84). Collagen content in total protein was lower in spring season (5.80 ± 2.44) as compared to autumn period (6.05 ± 0.78%), yet without statistically signifi cant diff erence (p = 0.73). Calculated energy value was higher in autumn (117.38 ± 13.09 kcal) than in spring (113.06 ± 11.27 kcal) without statistically signifi cant diff erence (p = 0.38). Th e diff erence in calculated energy values in spring and autumn periods is expected, having in mind higher fat content in carp fi llets sampled in October as compared to those sampled in April. According to the results reported by Yeganeh et al. (2012), fat and protein contents in samples of both "wild" carp and farmed carp decreased from summer to spring, whereas moisture contents increased during the same period. Over a 6-month research conducted between May and October, Swapna et al. (2010) did not establish any signifi cant seasonal variations in fat contents in fi sh meat. On the other hand, Guler et al. (2008) reported highest fat content in carp fi llets during winter months. Th is diff erence was explained by well-known seasonal variation in fat content associated with the changes of environmental temperature. According to the results obtained by Rasoarahona et al. (2004), fat content in carp muscle tissue was lower during warm months and higher during colder period of the year. It is to be emphasized that chemical parameters in wild fi sh are highly determined by the conditions of aquatic environment, which is well established source of nutritive matter. In farmed fi sh, diet containing commercially available feed-mixtures provides the abundance of nutrients thus determining the composition of meat. Protein content in meat is mainly determined by intrinsic factors such as species and size of the fi sh rather than by the diet itself. Fat content in the meat of farmed carp reported by Yeganeh et al. (2012) was in accordance with the results obtained in our study. With respect to protein contents determined in spring and autumn periods, Yeganeh et al. (2012) did not report any statistically signifi cant seasonal diff erences. Th e obtained results suggest a favorable ratio of nutritive matters in farmed carp, which could be attributed to high level of available nutrients in purifi ed slaughterhouse wastewater. Th e results obtained in our study are in compliance with the results reported by other authors, who have conducted a number of researches on carp meat quality in diverse production systems. Chemical composition of carp meat was closely similar to fi sh reared in semi-intensive farming system in a commercial fi shpond, where carps were fed concentrated feed mixtures and their meat had protein content of 17.30 ± 0.39% and fat content of 3.41 ± 1.37% (Ljubojević et al., 2013c). Such protein content is signifi cantly higher and fat content signifi cantly lower as compared to the cage-farmed carps of the same body weight, which were fed with corn. Th e protein and fat contents established in these carps were 16.23 ± 0.54% and 9.79%, respectively (Ljubojević et al., 2013c). A range of earlier researches suggested substantial variability in the quality of carp meat depending on the age, farming system and diet of the fi sh (Marković et al., 2016). Th e reported fat contents in carp meat ranged between 2.3 and 16.8%, while somewhat lower variability was observed with respect to protein contents, which was within a range from 14 to 18% ( Protein content in fi llets of carp from semi-intensive production system fed diet supplemented with corn and wheat reported by Ćirković et al. (2011) was 15.59%, whereas our results obtained in samples collected in spring and autumn season were 17.99% and 17.67%, respectively. Interestingly, significantly lower protein content was measured in fi llets sampled during autumn season (17.67%) as compared with the results obtained in spring sampling period (17.99%). Th is could be attributed to the eff ects of diet, that is, higher percentage of grains in fi sh diet in autumn period, having in mind that no edible off al were added in the last three months. Ljubojević et al. (2015) observed that decreased protein content in fi sh feed results in an increase in fat content and decrease in moisture and protein contents in fi sh muscle tissue. Increased amount of carbohydrates in carp diet leads to a more extensive deposition of visceral and muscle fat (Trbović et al., 2013). Th e fat content in fi sh meat varies according to the species, season of the year, water temperature and diet (Guler et al., 2008(Guler et al., , Ćirković et al., 2011. Elevated fat content in carp meat negatively aff ects its sensory characteristics because increased fat amount aff ects the meat consistency and texture.

CONCLUSION
Th e use of purifi ed slaughterhouse wastewater in carp fi sh-pond positively aff ected the production parameters and resulted in production of carp characterized by adequate meat quality corresponding with that of the carp reared in conventional production systems.
Th e results of this research and their comparison with earlier researches on the quality of carp meat strongly suggest that the use of purifi ed slaughterhouse wastewater results in a production of good-quality carp meat.
Th e use of slaughterhouse wastewater in fi sh production represents a novel approach to the sustainability of meat industry and environment protection. Th e application of this concept within a slaughterhouse is crucial in the aspect of environment protection, having in mind the requirements and standards aimed at minimizing the environment pollution imposed by EU. In that respect, such requirements have to be fulfi lled, and Serbian legislation on environment protection must be harmonized with the EU regulations.

Author's Contribution:
MP and DLJP made substantial contributions to basic idea, conception and design, acquisition of samples and data, analysis of the data and interpretation of results; NP, NN, NP and SVK was involved in draft ing of the manuscript, revising it critically for important intellectual content, and DLJP and MŽB gave the fi nal approval of the manuscript to be published.