Introduction

In the last few decades, there has been steady growth in aquaculture production globally. In Africa, the success story of aquaculture cannot be told without mentioning Clarias gariepinus (African catfish). It is the second most important fish cultured in Africa, (after Tilapia species).The highest annual production of C. gariepinus in Africa is from Nigeria (FAO, 2012). The choice of the African catfish for culture is dependent on many factors which includes; fast growth, high market price, ease of breeding and hardiness among others. However, being an omnivorous fish species, it requires high amount of crude protein in its feed, ranging between 35-42% depending on the age and size (Faturoti et. al., 1986). Hence the high nitrogenous waste in its culture water, since not more than 25% of the nitrogen in the feed is being utilized by the fish and the rest end up being a waste either as uneaten feed, fecal droppings or metabolic wastes (Piedrahita, 2003). High amount of wastes especially toxic inorganic nitrogenous substances (NH_4⁺ and NO_2^-), form part of the major water quality problems in intensive aquaculture systems (Avnimelech, 1999). There are different types of waste in fish culture systems but among them solid waste is the most dangerous and should be removed quickly and efficiently (Timmons and Summerfelt, 1997). This is because on its own it can clog the gills and suffocate the fish or if decomposed will increase both the total suspended solids and total dissolved solids, as well as the nitrogenous wastes compounds in the culture water. Losordo and Timmons (1994) also noted that when solid waste in aquaculture systems is broken down by bacteria within the system, fecal solids and uneaten feed will consume dissolved oxygen and generate ammonia–nitrogen. This may lead to increase in the biochemical oxygen demand (BOD) and chemical oxygen demand (COD) in the culture medium. The settleable wastes solids in culture water can be removed when accumulated, through properly placed drains at the bottom of the cultured tank, while the suspended solids are more difficult to remove. The usual method of removal is by flocculation through addition of coagulant or flocculation aids (AWWA, 1997). Raghuwanshi et al. (2002) defined flocculation as the process through which microfloc particles are brought together to form large agglomerations by mixing them together physically or through binding action of flocculants. Numerous substances has been used as coagulants in water treatments and this includes both synthetic and natural materials such as Aluminum sulphate (Alum), Ferric chloride, ferrous sulphate, lime, Zea mays, Moringa oleifera seed etc. (Ebeling et al., 2004). Though Alhassan (2008), stated that chemical coagulants such as Alum are not cost ineffective. Alum still remains the most commonly used coagulant in water treatments. Alum has the capacity to coagulate and trap solid matter which may be floating (Ebeling et al., 2004). This includes algae and some other organic materials that may be beneficial to the cultured fish. The primary disadvantage of Alum is that its effectiveness over a limited pH range of 6.5-7.5. Among the natural plants materials reported to have been used effectively as coagulants are Zea mays (Raghuwanshi et al., 2002) and Moringa oleifera seed powder (Akinwole and Jioke, 2006; Oluwalana et al., 2004). Akinwole and Jioke (2006) used Moringa oleifera seed powder as a coagulant to remove solid waste in aquaculture wastewater, the treated water was however not reused. The extensive literature also did not reveal any previous work where the treated wastewater with either alum or moringa seed powder was re-used for fish culture.  Therefore it may be necessary to ascertain effect of re-using the treated wastewater on the health status of fish. Pandey and Pandey (2001) asserted that blood is an important part of living organism and it can be used to determine the health status, because blood parameters changes as the organism respond to changes in its environment. Among the important haematological parameters are packed cell volume (PCV), white blood cell count (WBC), red blood cell count (RBC) and haemoglobin (Hb). Measurement of haematological parameters can be used to evaluate the physiological environment and husbandry stress in fishes (Dienye and Olumuji, 2014). In an effort to determine the effect of re-using treated wastewater on the health status of the fish, this study therefore examines the culture water quality parameters and haematological responses of Clarias gariepinus cultured in wastewater treated with Alum orMoringa oleifera seed powder.

1 Results

The results of the water quality parameters before and after treatment with the coagulation aids (Table 1). All treatments had an average temperature of 26˚C with only slight variations which was not significantly different. The control had the highest dissolved oxygen of 4.98±0.06 mg/l, alum treated water had dissolved oxygen of 3.69±0.04 mg/l while moringa treated water had the least dissolved oxygen of 3.69±0.09 mg/l. There was significant difference (p=0.001) between the control and both the alum and moringa treated water. But the difference between alum and moringa treated wastewater was not significant. The pH observed in the control was 7.40±0.04, alum treated water was 7.50±0.02 while the moringa seed powder treated water had a pH of 7.20±0.02.The difference in pH was only significant (P=0.003) between the control and moringa seed treated wastewater. There was no significant difference (P=0.952) in the ammonia-nitrogen concentration between the alum powder treated water and moringa seed powder treated water, 8.0±0.00 mg/land 7.5±0.28 mg/l respectively, but the control was significantly lower (P=0.002) in ammonia-nitrogen (0.13±0.02 mg/l) than both alum and moringa treated water. Nitrite-nitrogen and nitrate-nitrogen were not detected in both the control and alum treated water but only in moringa treated water, with values 0.02±0.01 mg/l and 0.42±0.23 mg/l respectively.

The total suspended solids in the alum treated wastewater were 962±180.33 mg/l while that of moringa was 745.17±233.16 mg/l and the control was 100.42±1.40 mg/l. There was significant difference (P=0.000) in the total suspended solids recorded among all the treatments with the control having the least TSS. Total dissolved solid also follow the same pattern with TSS, alum treated water has the highest TDS of 1 116.38±48.7 mg/l, followed by moringa treated water, 870.75±39.34 mg/l while the least, 180.42±3.58 was recorded in the control. The difference was significant (p=0.000) among all the treatments. There was significant difference (P=0.000) in the settled floc depth (cm), with moringa treated water having settled floc of 0.83±0.09 cm while alum treated water had 0.74±0.10 cm. In addition, Moringa seed showed a better performance in terms of solid removal, if the final mean value obtained is compared with the wastewater sourced for initial treatment. Moringa treated water has a 34% and 61% reduction in TSS and TDS respectively compared to 15% and 51% reduction obtained in alum treated water.

The haematological indices of fish reared in the control, alum powder treated wastewater and Moringa oliefera seed powder treated wastewater (Table 2). The packed cell volume (PCV) result showed that the fish cultured in alum treated water had the least PCV of 17.00±1.73% which was significantly different (P=0.018) from the PCV in fish cultured in the control (25.67±5.03%) and those culture in moringa treated wastewater (27.00±1.73%). Though PCV was also higher in fish culture in moringa treated water than that of the control but the difference was not significant (p=0.872).

The results revealed that white blood cell (WBC) was significantly higher ((P=0.033) for fish cultured in alum treated water (20 300.00±259.81 mm_^-3) compared to 12 933.33±3 010.54 mm_^-3 in the control and 14 950.00±3 377.50 mm^_-3 in moringa treated water.

Fish cultured in moringa treated water had the highest value of 2.58±0.55x10_³ mm_^-3 for red blood cell (RBC), followed by fish in control with, 2.18±1.04x10_³ mm_^-3 (with no significant difference between the two, P=0.095), but the two were significantly (P=0.018) higher than 1.14 ±0.01x10_³mm_^-3observed in fish cultured in alum treated water. The result obtained for haemoglobin (Hb) showed that fish cultured in moringa seed powder treated water had the highest haemoglobin level of 8.53±0.15 g/100 ml which was significantly different (P=0.033) from alum powder treated water with 5.37±0.12g/100ml but not significantly different (p= 0.974) from the control (8.37±1.60 g/100 ml). The highest platelet was recorded in fish cultured in alum powder treated water (266 666.67±37 527.77 mm_^-3), while the control had 168 333.33±62 947.07 mm_^-3, and fish cultured in moringa seed powder treated water had 177 666.67±89 645.60 mm_^-3. There was no significant difference (P=0.219) in the platelet among all the treatments.

The fish cultured in the control had the highest lymphocyte of 64.67±5.86%, fish cultured in moringa seed powder treated wastewater had 60.00±5.57%, while those in alum powder treated water had the least lymphocyte of 48.67±2.31%. There was no significant difference (P=0.507) between the control and moringa seed powder treated water, but there was significant difference (P=0.016) between alum powder treatment and the other treatments. The monocytes recorded for each treatment showed that there was no significant difference (P>0.05) among the fish cultured in the three treatments. There was also no significant difference (P=0.380) in the eosinophil among the treatments but there was significant difference (P=0.027) in the basophils recorded for fish cultured among all the treatments. The differences was also significant (P=0.013) for the heterophils among all the treatments. There were the notable clinical signs (laceration on both the skin and the tail) observed on the fish cultured in alum powder treated water (Figure 1).

2 Discussion

The mean temperature recorded for all the treatments was within the range (25-32˚C) recommended for warm water fish culture (Dauda and Akinwole, 2014; Jenyo-Oni et al., 2010; Akinwole, 2005).The dissolved oxygen concentrations in all the treatments were within the desired range (3-10 mg/l) recommended for African catfish culture (Akinwole, 2005; Boyd, 1990). The pH range (7.0-7.2) recorded for all the treatments in this study was normal for Clarias gariepinus culture (Akinwole, 2005). Though the ammonia-nitrogen was far higher than the values reported by most researchers (Dauda and Akinwole, 2014; Al-hafedh et al., 2003) for culturing warm water fishes in water reuse systems but it was still below the safe limit reported by Akinwole (2005).The presence of Nitrite and nitrate indicated there was nitrification process in moringa treated water due probably to the possibilities of moringa encouraging the growth of nitrifying bacteria. Moreover, the value obtain for nitrite-nitrogen and nitrate-nitrogen are not harmful to warm water fish cultured in water reuse systems (Dauda et al., 2014; Al-hafedh et al., 2003). The two coagulating aids reduced the TSS in the wastewater but moringa seed powder performed better than alum. The two of them were unable to reduce the TSS effectively considering significantly lower value recorded in the control, which was the only one that fell within the range recommended for fish culture (Ajani et al., 2011). However the TSS value recorded in moringa treated water was below the value reported by Green et al. (2014), without reporting any negative effect on the growth performance of channel catfish culture in biofloc system. The amount of settled floc obtained in this research is similar to that of Akinwole and Jioke (2006).

Haematological parameters are routinely used for the evaluation of physiological environment and husbandry stressors in fishes (Dienye and Olumuji, 2014). The change in the blood characteristics of C. gariepinus as a result of stress due to exposure to toxic materials or pollutants, diseases or pathogens have been reported by a number of researchers (Owolabi, 2011; Douglas and Jane, 2010; Sotolu and Faturoti, 2009). Blood is an important component of living things and inevitable part of the immune system, changes in blood parameters can be due to the response of the organism to the changes in its environment. Hence, blood can be used to screen the health status of the fish that is exposed to toxicant or degraded environmental conditions (Pandey and Pandey, 2001). PCV recorded in both fish cultured in control and that of moringa treated water were within the range reported by Sotolu and Faturoti (2009). Lower value of PCV recorded in fish cultured in alum treated wastewater showed that the fish are not as healthy as other treatments (Sotolu, 2010) and the values reported fell within the values for fish said to be in anaemic condition (Owolabi, 2011). The increase in the PCV in moringa seed powder treated wastewater is similar to what was reported by Bello and Nzeh (2013) and Dienye and Olumuji (2014), they reported that there was increase in PCV in Clarias gariepinus fed varying level of Moringa oleifera leaf meal diet and also that the higher the PCV level, the more healthy the fish is while the lower the PCV the more susceptible the fish will be to infection. Also, the result obtained in the study was also in line with that of Ayotunde et al. (2011) who reported that there was significant increase in PCV of tilapia reared in different concentrations of Moringa oleifera seed powder, though Moringa oleifera application in this experiment was not fed to the fish. The decrease observed in the level of PCV in alum powder treated wastewater might be as a result of blood loss through the barbells, skin and tail laceration which was observed during the course of the study.

The white blood cells (leukocytes) are important components of the immune system, and they are concerned with defending the cell against invasion by foreign bodies. A high count of leukocytes is usually an indication of response to stress in animals (Davis et al., 2008). The WBC recorded in the control and moringa seed powder treated wastewater was within the permissible limit and is similar to what was reported by Dienye and Olumuji (2014). White blood cells (WBC) and lymphocytes are the defense cells of the body. Lymphocytes are non-granular leukocytes that are responsible for the release of antibodies that engulf the foreign bodies. Heterophils on the other hand are granulocytes, and they are usually the first responders to microbial infections. Douglass and Janes (2010) reported that the amount has implication on immune responses and the ability of the animal to fight infection. High WBC count was noted to have relationship with microbial infection, or the presence of foreign body or antigen in the circulating system (Oyawoye and Ogunkunle, 1998), this might account for the high WBC count and high mortality rate recorded in the fish cultured in alum powder treated wastewater.

The RBC counts in the control and alum powder treated wastewater in this study were fairly comparable with the range 1.70x10_⁶ mm_^-3-4.00x10_⁶ mm_^-3reported by Bhasker and Rao (1990). However, the decrease in RBC in alum powder treated wastewater may be ascribed to the presence of microbial infection or foreign bodies in the culture system. The excess secretion of mucus observed in alum powder treated wastewater might be accountable for their susceptibility to microbial infection. Haemoglobin (Hb) range (7.90-8.90 g/100 ml) recorded in this study compared well with 8.70 g/100 ml reported for Clarias gariepinus by Sowunmi (2003). The reduction in the Hb concentration in alum powder treated wastewater could imply that there is poor transportation of oxygen from the respiratory organs to the peripheral tissue (Robert et al., 2000).The findings of the study indicated that solids removal in wastewater with alum though resulted in good water quality parameters that is suitable for fish culture, the hematological studies revealed negative effect on the health and immune responses of the fish cultured. The use of M. oleifera seed powder has both advantages of improving the water quality and improving the health status of the fish.

3 Materials and Methods

The experiment was carried out in the teaching and research fish farm of the Department of Aquaculture and Fisheries Management, University of Ibadan, Ibadan, Nigeria. The design was a complete randomized design comprising three treatments which includes; Treatment 1, the control where borehole water in the farm was used for fish culture, Treatment 2; Alum treated wastewater was used for fish culture and Treatment 3; Moringa oleifera treated wastewater was used for fish culture.

3.1 Flocculation process

The two coagulation aids used in the study are Aluminum sulphate (Al₂[SO₄]₃) called Alum, which is a synthetic coagulation aid and Moringa oleifera seed, a plant based natural coagulation aid. The Alum was obtained from the University of Ibadan water treatment plant while Moringa oleifera seed was obtained by hand picking from the Moringa oleifera tree in the Department of Forest Resources Management, University of Ibadan. Dried and matured Moringa oleifera seeds were collected, the seeds were removed from the pods and sun dried. The dried seeds were later crushed and grounded with mortar and pestle till a fine particle was obtained, and sieved using a 0.15 mm mesh size netting in order to get a fine powder. The alum was also crushed and grounded, and later sieved using a 0.15 mm mesh sized netting in order to obtain a fine powder. Initial effluent water sample used was collected from an earthen pond stocked with African catfish (Clarias gariepinus) grow out system in the University of Ibadan fish farm (Details of the water quality parameters is provided along with the results) (Table 1). A total of 140 litres wastewater was obtained from the culture pond (70 litres for each of the coagulating aids) in the first week while the subsequent wastewater was obtained from the culture experiment itself in order to fulfill the primary objective of testing the reuse potential of the treated wastewater. The collected wastewater sample was subjected to the flocculation.

The flocculation process was in three stages. First, the coagulation aids were weighed with the use of an electric weighing balance and added at 60 mg/l for Alum treated wastewater (Treatment 2), 120 mg/L for M. oleifera treated wastewater (Treatment 3) (Though equal concentration was initially projected for the two coagulation aids but Alum was changed to 60 mg/L after 4th week due to high mortality recorded in the tanks using alum treated wastewater). The water was then subjected to manual mixing with a turning stick at the rate of 100 revolutions. The average time taken for mixing was determined by recording the period elapsed to have 100 revolutions with the use of a stopwatch. The flocculated water sample was allowed to settle for 24 hours. The supernatant was carefully decanted into another plastic container and then poured into the fish rearing tanks for each treatment. Each treatment has three replicates. Circular plastic tanks were used for the experiment. Each circular tank had a diameter of 45 cm, radius 22.5 cm and a depth of 29 cm. The water was maintained at the depth of 15 cm, in order to ensure 20 litres of culture water in each tank. Ninety Clarias gariepinus juveniles with the mean weight of 10±0.00 g/fish were stocked at 10 fish per tank. A total of nine tanks were used for the two treatments and the control, and each treatment has three replicates. The fish stocked were fed twice a day at 5% body weight with 40% crude protein 2 mm commercial feed for the first four weeks, while for the remaining weeks 3 mm commercial feed was administered based on the weight gained at 5% body weight. The culture water was drained completely and replaced every 72 hours with newly treated wastewater.

3.2 Assessment of water quality parameters

Selected water quality parameters of the wastewater were measured before treatment and after treatment with the coagulation aids. The pH, ammonia-nitrogen, nitrite-nitrogen and nitrate-nitrogen were determined using API_^® fresh water test kit. The temperature (°C) in the culture tanks was measured every 72 hours with the use of mercury -in -glass thermometer as described by Dauda and Akinwole (2014).The dissolved oxygen was determined through the use of Labtech_^® dissolved oxygen meter. The total dissolved solid (TDS) and total suspended solid (TSS) were evaluated by gravimetric methods in line with APHA (2012).

3.3 Assessment of haematological parameters

The haematological parameters were assessed before stocking and at the end of the experiment in order to examine the effect of the treatments aids on the health and well-being of the cultured fish. Blood samples were collected through the caudal peduncle as described by Sotolu (2010). The haematological parameters measured were: total erythrocyte count (RBC), total white blood cell count (WBC), hematocrit (PCV), haemoglobin concentration (Hb), platelet, lymphocytes, monocytes, eosinophils and basophils. Measurement of PCV and Hb concentration were done in line with methods of Dacie and Lewis (2001), Determination of RBC and WBC were  done manually using Neubauer haematocytomer (Dacie and Lewis, 2001), while other haematological parameters were measured following the protocol of Tiez (1995).

3.4 Statistical analysis

The mean values for each water quality and haematology parameter were calculated using descriptive statistics (mean and standard deviations). The differences in mean value for each parameter among the three treatments was evaluated using one way analysis of variance and where a significant difference was observed, Turkey’s HSD range test was used as follow up to determine the specific pair that were statistically different at p<0.05 through the use of IBM SPSS 21 software.

Authors’ contribution

The idea of the research work was conceived by the lead researcher (Akinwole A.O.), the design and planning was done by all the authors, with the second author (Dauda A.B.) given the primary responsibility of the research design. All the authors worked together for the experimental set-up. The routine work of daily feeding was done mostly by the third author (Ololade O.A.). The weekly water quality parameters and fish growth assessment was done together by all the authors, with the second author doing the coordination. Analysis of the data was led by the lead author, while the third author prepared the draft for methodology. The second author prepared the draft for literature review and all the author participated in results and discussion writing. The paper was also jointly agreed upon to be sent to your reputable journal and there is no conflict of interest among the researchers.

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