Mapping of physical-chemical, microbiological, and chemical component characteristics of water samples from Nile tilapia slaughterhouses

The aim of this study was to analyze the physicochemical and microbiological characteristics of the water used in fish processing and tilapia fillets in slaughterhouses. The study analyzed the processing water from nine slaughterhouses. The water samples for analysis were collected at three points: process water (PW), it is the water used inside the slaughterhouses in direct contact with the fish; clean water entering the purification tank (EPT) and water leaving the purification tank (LPT). The processing waters of the nine tilapia slaughterhouses were analyzed and characterized according to their microbiological and physical-chemical characteristics. The results of microbiological analyzes meet the values indicated by legislation, in most cases. Regarding microbiological data, we can highlight that there was an increase in the total coliforms of the water entering the purification tank to the water leaving the purification tank in five slaughterhouses. Still, there was an increase in the aerobic mesophilic bacteria content observed in the outgoing water in relation to the inlet water of the purification tank in seven slaughterhouses. In relation to the physical chemical analyzes for the process water samples, the results show that the evaluated indices are in accordance with the values indicated by the legislation. The levels of Cd, Mg, Sc and Cd were below that detectable by the analysis in all Research, Society and Development, v. 10, n. 11, e164101119066, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19066 2 slaughterhouses. The levels of Al, Sc, As, Rb, Ba, Pb, Mn, As, Se, Rb, Ag, Sb, Ba and Pb were detected only in one or two slaughterhouses. It is concluded that although some abattoirs have water characteristics outside the limits indicated by the legislation, the observed changes were not significant and small adjustments are necessary for the adequacy.


Introduction
Among foods of animal origin, fish are present the greatest conditions favorable to deterioration, and, therefore, are considered highly perishable. This is mainly since the fish have a pH close to neutrality, a high amount of nutrients and water content, which means that a wide range of biochemical reactions can be triggered, facilitating the entry and development of microorganisms (Gonçalves, 2011).
Thus, the control of water from the creation of the fish to the final product of the fish is essential for maintaining a low range of microorganisms present and of metal residues and other chemical elements in the meat of the fish at the end of the entire process. The large number of bacteria found in mucus, gills, and intestines of fish (González, et al., 1999) makes microbial contamination the most important concern in fish processing (Gram & Huss, 1996). These changes in association with intrinsic and extrinsic factors can increase the susceptibility to deterioration of the fish, directly influencing the original Research, Society andDevelopment, v. 10, n. 11, e164101119066, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19066 3 organoleptic characteristics of the fish, facilitating the development of food-borne diseases (Huss, 1997;Massaguer, 2005;Jay, 2005, Boari, et al., 2008Gonçalves, 2011), and compromise the product's useful life (Adebayo-Tayo, et al., 2012). As far as points of interest for microbial control in a slaughterhouse, the water in the purification tank, the processing of the fish at the time of processing and the water used in the ice used for the maintenance of the chilled or frozen fish, are the points that deserve attention and care.
In addition, the wastewater from both the fish purification and the water used in fish processing must be monitored, as the disposal of water without proper care and with some contamination, can harm the environment, being a potential focus of eutrophication of natural waters and artesian wells located close to the slaughterhouse. The control and regulation of the water quality used in all stages of fish processing is of paramount importance both to ensure food security and to minimize the possible environmental damage that can occur due to improper disposal and contaminated water to the environment.
Thus, the study was carried out with the objective of analyzing the microbiological and chemical characteristics of the water used in processing fish, in Nile tilapia slaughterhouses.

Study Area and sampling
The study was carried out in five cities in the western region of the state of Paraná, Brazil ( Figure 1). The area of this study comprises the largest developed region in the Nile tilapia cultivation and slaughter segment in southern Brazil. Nine slaughterhouses were chosen to assess the quality of the water used in the animal purification and slaughtering internal processes, as well as to evaluate the quality in terms of physiochemical and microbiological aspects of the fillets produced. The slaughterhouses were identified as S1, S2, S3, 8 S4, S5, S6, S7, S8, and S9. A collection of samples of fillets and water was carried out from December to March. The collections followed methodologies for sampling as recommended by ICMSF  Research, Society andDevelopment, v. 10, n. 11, e164101119066, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19066 4 The water samples were identified as process water (PW), that is, it is the water used inside the slaughterhouses in direct contact with the fish; clean water entering the purification tank (EPT) and water leaving the purification tank (LPT) ( Figure 2). All identifications were followed by the number of the slaughterhouses evaluated. Source: Prepared by the authors (2020).

Microbiological analysis
Aerobic mesophilic and psychotropic microorganism analyses were performed using a representative part of the samples (25 g or mL), where they were stored in the Stomacher bag and homogenized for 60 seconds in a Stomacher machine with 225 g of chilled peptone saline diluent (0.85 % NaCl with peptone a 0.1 %). Followed by an appropriate 10-fold dilution of the homogenate using saline peptone as the diluent. For each blank dilution, replicates were prepared. 0.1 mL of each appropriate dilution step was spread on the surface of dry media in Petri dishes. Mesophilic bacteria were determined by using deep plate count agar (35ºC, 48 h) and psychotropic bacteria using surface plate count agar (20ºC, 120 h). Procedures prescribed by ICMSF (2002); Alimentarius (2007), and APHA (2017).
Total coliforms and thermotolerant coliforms bacteriological tests were performed after sampling. The methods used to recover the bacterial load were the most likely number (NLP) with the presumptive test in tryptose with lauryl sulfate at 35ºC for 24-48 h and confirmation in bright bile green broth at 2% (35ºC, 24-48 h). For coliform bacteria and for thermotolerant coliforms, confirmation in CE broth and incubation at 44.5ºC for 24 h was conducted (ICMSF, 2002;APHA, 2017).  For Salmonella spp., pre-enrichment by incubating the sample dilutions at 37ºC for 24 h was performed.
The elementary chemical concentration of the water and fillet samples was analyzed using a portable benchtop TXRF instrument, featuring an air-cooled low power X-ray metal-ceramic tube with a molybdenum target, working at 20 keV of energy for 1000 s, and a liquid nitrogen-free Silicon Drift Detector (SSD) with ranging element from sodium (Na) to uranium (U) (Bruker, 2007). For this particular research, the following elements were analyzed: Na, K, Mg, Ca, Ba, Sc, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Nb, Ag, Cd, Pb, P, S, Cl, As, Se, and Br.

Results
Next, the results obtained for the microbiological and physicochemical parameters of the water will be presented and discussed. Table 1 presents the results regarding the microbiological analysis of the water samples from the entrances and exits of the fish purification tanks and the water samples used in the tilapia slaughter process. For total coliforms (TC), values ranging from <1.10 to> 23 MPN mL -1 were observed in the water inlet of the purification tank, from 5.10 to> 23 MPN mL -1 in the water out of the purification tank, and < 1.10 MPN mL -1 in the water used in the fish processing process in all slaughterhouses evaluated in this study. There was an increase in the TC of the water entering the purification tank to the water leaving the purification tank in slaughterhouses S3, S5, S6, S7 and S9.

Microbiological parameters of process water
In the analysis of thermotolerant coliforms (TCL), values <1.10 were observed in the inlet water of the tanks, with the exception of slaughterhouses S3 and S8, which presented values of 5.10 MPN mL -1 . In the water leaving the purification tank, all slaughterhouses showed values >23 MPN mL -1 of TCL, while in the process water, the value was <1.10 in all slaughterhouses.
Regarding the parameters of S. aureus and Salmonella ssp., all samples expressed values of <10 CFU mL -1 , respectively.
The content of aerobic mesophilic bacteria (MAB) varied from <1.00 to 4.75 CFU mL -1 in the water entering the purification tank, while in the water leaving the purification tank the values ranged from 2.13 to 5.45 CFU mL -1 . An increase in the MAB content was observed in the outgoing water in relation to the inlet water of the purification tank in all slaughterhouses, with the exception of S3 and S9. For process water, a value of <10 was observed in all slaughterhouses, with the exception of S9 slaughterhouse, which had an exact value of 4.56 CFU mL -1 . In addition, a significant difference was observed for the MAB content obtained in the water from the purification tank between the slaughterhouses evaluated, where S8 had the highest value and differed from the others, and S5 had the lowest value.
Regarding psychotropic bacteria (PB), with the exception of the S9 slaughterhouse (4.68), all presented results <1.00 (Log CFU mL -1 ) in the water entering the purification tanks. In the water leaving the purification tanks, he observed results ranging from 2.97 to 4.65 (Log CFU mL -1 ), with the S6 slaughterhouse showing the highest value (4.65). For the water used in fish processing, a value of <10 was observed for all slaughterhouses, with the exception of S9, which presented a value of 5.68 (Log CFU mL -1 ). Table 2 presents the results regarding the physical-chemical analysis of the water samples from the entrances and exits of the fish purification tanks and the water samples used in the process of processing the fish.

Chemical Analysis
The results presented for total dissolved solids (TDS) in the inlet water of the purification tank and in the water used in the process, showed values lower than <0.10 mg L -1 . On the other hand, at the exit of the purification tank, this variable showed values from 80 to 2137 mg L -1 , with slaughterhouse S8 having the lowest value and S2 the highest value.
For the data obtained from the sedimentable solids (SS) (mg L -1 ), it was observed both in the water in and out of the purification tank, and in the process water, a value below 1 mg L -1 .
The results of the alkalinity (mg L -1 ) of the inlet water of the purification tank ranged from 2.61 in the S2 slaughterhouse to 37.50 in the S5. For the water leaving the purification tank, the alkalinity results ranged from 3.03 in S1 to 37.80 in S5. In the water used in fish processing, the variation was between 10.40 in S1 to 31.70 in S6.
The nitrogen (N) (mg L -1 ) content observed in the inlet water of the purification tank and in the water used in the processing of fish was <1.00, while, in the water out of the purification tank values ranging from 0.35 to 0.95 were observed.
For the Biochemical oxygen demand (BOD) variable (mg L -1 ), values below 2 were observed for the inlet water of the purification tank and the fish processing water, while for the outlet water of the purification, values ranging from 25 to 1470 were observed.
For the evaluation of the chemical oxygen demand (COD) (mg L -1 ), it was observed that the water of entry in the purification tank and the water used in the processing of the fish presented values below 2.00, while in the water of disposal of the purification tank the values varied from 156 (S9) to 3270 (S1).
For the water used in fish processing, the content of free residual chlorine (FRC) was also analyzed, as the water is treated with chlorine. Values ranging from 0.18 (S9) to 1.62 (S8) were observed.

Discussion
The control of water quality for the handling of fish-based products is of great importance, since fish meat and its derivatives are excellent substrates for the development of microorganisms, including those of water transmission. All water that comes into contact with food must comply with the same microbiological standards as water for human consumption (Frazier & Westhoff, 2003).
In the food industry, water is essential and has high use and consumption, due to the various functions it plays in the processes and the need to sanitize establishments and equipment to guarantee the hygienic-sanitary quality of the final product (Massoud, et al., 2010). According to the World Health Organization (WHO) described in chapter 13 of the compiled manual Water Quality: Guidelines, Standards and Health (Ashbolt, et al., 2001) the microorganisms traditionally used to assess and monitor water quality belong to three classes, microorganisms that indicate sanitary deficiency, composed of heterotrophic bacteria and total coliforms; indicators of fecal contamination, which are part of the intestinal microbiota of man and warmblooded animals, the main one being E. coli; also pathogenic microorganisms such as Salmonella, Staphylococcus Aureus, among others.
The fish purification tank in a slaughterhouse, is used to clean the fish that could be stored in polluted water, as well as to eliminate the contaminants contained in the intestinal tract of the animals that could interfere in the flavor and quality of the fish for human consumption. Studies show that tilapia submitted to a purification process for 8 h, show a significant improvement in quality and flavor, and the purification process during this time is capable of removing the earthy flavor from the fillet, and thus, increasing its acceptability of significantly by consumers (Rohani, et al., 2009).
Making the level of harmful contaminants and microorganisms after purification undetectable or non-existent, favoring human consumption and food security. Likewise, the water used during the process for bleeding, evisceration, and filleting of the fish must be clean and adequate, so that there is no contamination of the fish meat. In this sense, thermotolerant coliforms are the first-choice microorganisms for the assessment of pollution of fecal origin in the environment and in water, since they are predominantly constituted by the bacterium Escherichia coli, currently considered the most suitable indicator (Garcia-Armisen, et al., 2007).
Regarding the number of microorganisms present in the purification water (please see Table 1A, and 1B), there is no specific citation in legislation. However, animals must undergo procedures capable of cleaning and removing dirt, respecting the particularities of the species, before entering the slaughter process (Brasil, 2018). The levels observed in the microbiological analysis in these samples are higher, possibly due to the presence of transport water and the emptying of the animals' gastrointestinal tract in the purification tanks.
The increase in the content of total coliforms and of thermotolerant coliforms in the water collected from the purification tank outlet in relation to the inlet water, shows the importance of the fish purification process, since all this microbial load that remained in the outlet water of the purification, was in the fish itself, and was eliminated before the slaughter process.
The analyzed water samples (Table 1C), which come into direct contact with fish derivatives in the industrial process, are within the parameters established by the legislation (ANVISA, 2011), only the S9 slaughterhouse presented results above 2.7 (Log CFU mL -1 ) for MAB (4.56) and PB (5.68). Although, not being high values, the presence of these microorganisms above the recommended by the legislation, are already an indication of inappropriate water for the intended use. It is necessary to take actions that aim to reduce or totally remove the presence of these microorganisms, because values above what is allowed by the legislation must be investigated to identify the irregularity and take steps to reestablish these parameters within the appropriate range (ANVISA, 2011). According to Frazier and Westhoff (2003), all water that comes into contact with food must comply with the same microbiological standards as water for human consumption. For this reason, the control and monitoring of the characteristics of the water used in the fish processing is of paramount importance.
The chemical parameters of the water are the most important for characterizing the quality, as it allows the classification through its mineral content, determination of the degree of contamination and concentration of toxic pollutants or excess of some metal.

Conclusion
Thus, it is concluded that there is a similarity between the analyzes carried out in the slaughterhouses, in relation to the physical-chemical, microbiological, and component quality, with few points capable of influencing its quality, which can be corrected with training and improvement of techniques.
The information obtained in our research is extremely important for the evaluation of the characteristics of the water used in tilapia slaughterhouses. We suggest that future studies carry out, together with the evaluation of the physical-chemical, chemical and microbiological characteristics of the water, the evaluation of fish meat and processed fillet, to relate the effects of any problems in water quality in fish meat.