Rthindrnth Mndl, Arind Ds, Denryn Chttopdhyy, Ajml Hussn,Suhendu Adhikri, Bidynth Pul, Frhn Hoque, Prthprtim Chkrrti,Bindu R. Pilli

aRegional Research Centre, ICAR-Central Institute of Freshwater Aquaculture, Rahara, Kolkata, 700118, West Bengal, India

bICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, 751002, Odisha, India

ABSTRACT

The problem of sewage disposal has received great attention worldwide. The raw sewage contains a variety of high inorganic and organic matters that affect natural water environment. To mitigate such problem, sewage may be recycled through aquaculture practice. Sewage recycling in aquaculture enhances water productivity through nutrients input. Proper loading of sewage ensures viable aquaculture; otherwise,fish mortality occurs due to poor water quality. To optimize sewage application, two different experiments were conducted, each with four treatments. In both experiments, three fish species namely rohu (Labeo rohita Hamilton, 1822), mrigal(Cirrhinus mrigala Hamilton, 1822), and bata (Labeo bata Hamilton, 1822) were tested in triplicate in FRP (Fibre-Reinforced Plastic) tanks. Different sewage concentrations (0, 25%, 50% and 75%) used in first experiment were prepared by mixing freshwater, showing Biochemical Oxygen Demand (BOD) 2.0 ± 0.4 mg/L, 10.8 ± 1.4 mg/L,19.6 ± 1.5 mg/L and 41.6 ± 2.58 mg/L, respectively. After 30 days rearing, results showed ≥75% fish survival in sewage concentrations up to 50% with BOD level 19.6 ±1.5 mg/L. Less than 50% fish survived in 75% sewage concentration, with BOD level as 41.6 ±2.58 mg/L. The second experiment was conducted for 90 days considering 50% sewage concentration as basal dose with BOD level as 19.6 mg/L as an acceptable limit for fish survival. Split doses of sewage were applied in T1, T2 and T3 treatments fortnightly, weekly and semi-weekly intervals, while single dose was used in C (control) treatment. Application of split doses resulted better hydrobiological changes, including nutrients recovery, in T1, T2 and T3 than that of single dose in control. Fish growth plotted with net primary productivity (NPP), phytoplankton and zooplankton densities exhibited positive correlations in T2 (12 doses) and T3 (24 doses), considered as optimal doses to ensure better water productivity for desirable fish production than sewage with single dose or limited doses (6 doses).

ARTICLEINFO

Keywords:

Sewage

Split dose

Water productivity

Fish rearing

1.Introduction

The disposal of sewage which is a worldwide problem has received great attention. The raw sewage contains high inorganic and organic compounds, soaps, synthetic detergents, acids, phenyls, petroleum products, industrial effluents, several chemicals of agricultural uses and many other products used in our modern society. Biotic organisms such as harmful bacteria, virus, worms and protozoa remain in high density in sewage that affects natural water environment (Lahiri, Ghosh, &Sarkar-Paria, 2018). Nevertheless, sewage water is rich source of nutrients. It has been estimated that the quantity of nitrogen, phosphorus and potassium from annual discharge of sewage from several towns and cities of India are respectively 90, 32 and 55 metric tons valued Rs. 6.10 crores (1$ =INR 69.00) (Chakrabarti, Ghosh, Mukhopadhyay, &Jayasankar, 2011). This huge amount of nutrients may be recovered through effective procedure of sewage application through aquaculture.

Domestic sewage has been utilized for fish production in farmers’field since decades (Edwards, 2008). The previous attempts on the utilization of sewage for aquaculture through effective system of its treatments and method of application in ponds have been considered as means of nutrients harvest, effective recycling of used water, water conservation and reducing pollution load in natural water bodies (Al Baz, Otterpohl, & Wendland, 2008; Bunting, 2007; Bunting & Edwards,2018; Bunting, Pretty, & Edwards, 2010; Dasgupta, Pandey, Sarangi, &Mukhopadhyay, 2008; Datta, Roy, & Saha, 2000; Edwards, 2008;Ghosh, 1990; Grant et al., 2012; Jana, 1998; Jana, Heeb, & Das, 2018;Mandal, Chakrabarti, & Jayasankar, 2015; Mandal et al., 2018). However, most of the aquaculture practices by sewage application have been in traditional ways which usually encounters certain problems like excessive growth of phytoplankton leading to eutrophication, disease occurrence, dense population of microflora, poor water quality,fish mortality and low fish yield (Bhakta & Jana, 2006; Bhowmik, Chakraborty, & Chattopadhyay, 2000; Buchauer, 2007; Das, 2018; Das, Das,Ghosh, & Bhowmick, 1994; Jana & Datta, 1996; Lahiri, Sarkar-Paria, &Jana, 2015; Mara, 2008; WHO, 2008). Such problems are largely attributed to indiscriminate application of sewage in ponds without considering its quantity and quality, particularly nutrients and BOD level of sewage required. Sewage, by and large, has an excess amount of nutrients and organic matters: total solids, 720 mg/L; BOD, 400 mg/L;COD, 430–560 mg/L; organic carbon, 43–47 mg/L; PO, 08 mg/L; and total nitrogen, 25 mg/L (Lahiri et al., 2018; Mandal et al., 2015). The application of such sewage may cause hazard to the aquatic biota,disturb ecology of culture water and thereby, damage aquaculture practice.

Evidently, in and around Kolkata City (India), 100 L of domestic sewage are released per person day(Jana, 1998). Recently, Ghosh(2018) mentioned that in Kolkata metro for every 115.6 L of potable water is supplied to households, of which 92.5 L becomes sewage water considered to be wastewater. Sewage water released from Kolkata metro to East Kolkata Wetlands (EKW) accounts 1300 MLD (Million Liter Day), of which 320 MLD is available for aquaculture covering 5852.14 ha (Ghosh, 2018). Such a huge amount of untreated sewage water damages ecology of culture water that eventually causes fish mortality and poor amount offish yields. Intake of proper amounts of sewage water into aquaculture is essentially required to maintain BOD level and therefore, sewage water needs to be properly managed.Considering the above problems, the present study was undertaken with the following objectives: (i) determination offish survival at different sewage concentrations, with conducive limit of BOD level (ii)finding out effective split doses of sewage application in nutrients recovery for water productivity and (iii) evaluation of water qualities and microbial load, suitable for fish rearing.

2.Materials and methods

2.1.Sewage effluent

Sewage effluent which was used for this experiment emanated from domestic source and then underwent mechanical treatment by KMWSA(Kolkata Metropolitan Water Sanitation Authority) at Titagarh. An amount of Primary Treated Domestic Sewage Effluent (PTDSE) of Titagarh reached Regional Research Centre (RRC), Central Institute of Freshwater Aquaculture (CIFA), Rahara through 1.5 km long pipeline by gravity. It was kept storing in cemented pit inside the farm premises and allowed to have natural suspension for 5 days before its use in the experiments. In the pit, Biochemical Oxygen Demand (BOD) of supernatant effluent of PTDSE was measured as ranging between 50 and 60 mg/L, which was considered as 100% concentration as used in this experiment. Other chemical parameters of supernatant effluent were measured as in the range: pH, 7–7.5; Dissolved Oxygen (DO), 0.5–1.5 mg/L; Total Alkalinity, 250–340 mg/L;Total Ammonia Nitrogen (TAN,NH–N),2.2–3.5 mg/L; and Phosphate (PO) 1.5–3.0 mg/L.

2.2.Experimental design

Two experiments were conducted separately in FRP (Fiberglass Reinforced Plastics) tanks, each with 1000 L capacity. First experiment was established to test tolerance limit offish for 30 days. The PTDSE(100% concentration) was diluted with freshwater and made three different treatments such as 25%, 50% and 75% concentrations and a control with freshwater stored in a pond filled up by a tube well, each treatment had three replicates. In each tank, 800 L was established as an experimental volume with 0.6 m depth.

The second experiment was established to evaluate dose-response of sewage effluent in relation to water productivity and fish growth for 90 days, after considering 50% concentration of effluent (PTDSE) as acceptable to fish survival observed in the first experiment. Total four treatments were established, each with three replicates. Control (C)tanks were filled with 800 L of sewage and freshwater in 1:1 (v:v) ratio.For other three treatments (nine tanks), each tank was filled with 700 L of freshwater and sewage effluent (PTDSE) with 4:3 ratio (400 L freshwater +300 L effluent). To make up the total water volume at 800 L in these tanks, the remaining 100 L of effluent (100% conc.) was added as post stocking applications in 6 (fortnight for T), 12 (weekly for T) and 24 (semi-weekly for T) equal split doses in respective treatments till 90 days.

2.3.Operational details

Each tank, after being filled with required volume, was left for 5 days for stabilization. Bottom of each tank was provided with 1.0 inch soil base. In both 1st and 2nd experiments, twenty four fish comprising three common cultivable carp species as rohu (L. rohita), mrigal (C. mrigala)and bata (L. bata) were randomly released in each experimental tank,with 1:1:1 ratio. Initial mean body weight of rohu, mrigal and bata were 16.5 g, 12.5 g and 11.24 g in the 1st experiment, and 19.3 g, 13.5 g and 12.3 g in the 2nd experiment.

2.4.Analyses of hydro-biological parameters

Temperature was recorded at 12:00p.m. and pH was measured with digital instrument (Eutech, pH Totur, CyberScan). However, some selected parameters such as DO, BOD, total alkalinity, TAN (NH–N) and phosphate (PO) were estimated using following methods:

2.4.1.DO (Dissolved oxygen)

Dissolved oxygen of water samples was estimated following the Winkler’s method (APHA, 1998). Water sample was collected in a 300 mL BOD sampling bottle avoiding any air bubbles, then 1 mL of manganous sulphate solution followed by 1 mL of alkali iodide-azide reagent were added. When precipitate settled sufficiently to a level, 1 mL of concentrated sulphuric acid (HSO) was added to dissolve the precipitate and the color changed to golden yellow. Sample of 50 mL was taken in a conical flask and few drops of starch indicator were added to it till the color turned dark blue. Then the sample was titrated against N/40 sodium thiosulphate till the sample became colourless. The volume of the titrant was recorded and the result was expressed as mg/L.Dissolved oxygen content was calculated using following formula:

2.4.2.BOD (Biological Oxygen Demand)

Initially, 60 ml of sample was collected from culture water and made 600 ml by 10 times dilution. Then entire diluted sample was churned for 1.0 min so as to addition of Ointo the diluted sample. The 600 ml sample was put into two 300 ml BOD sampling bottles separately, each contains 300 ml. Out of two bottles, DO was measured from one bottle as the method mentioned earlier. Another bottle was kept into BOD incubator for 5 day at 20C or below. After 5 days over, its DO was measured as the method mentioned. BOD was measured from the difference of initial bottle and incubated bottle using following formula (APHA,1998);

2.4.3.Total alkalinity

Total alkalinity was calculated through titration method (APHA,1998). Water sample (50 ml) was taken in a conical flask, and 2–3 drops of phenolphthalein indicator were added to it, but no pink color developed as pH value of all water samples were below 8.3. To the same flask 2–3 drops of methyl orange indicator was added and sample was titrated against 0.02 N HSOtill yellow color changed to orange. The total burette reading was noted down and result was expressed as mg/L.Total alkalinity was calculated using following formula:

2.4.4.Plankton assessment

Phytoplankton and zooplankton were collected by filtering 10 L of culture water from each tank through plankton net having mesh size 70 μm. Collected water of each tank measuring about 7.0 mL was then put into an another measuring tube and diluted up to 10.0 mL. Of which 1.0 mL was put into Sedge-wick Rafter plankton counting cell under a compound microscope and counted manually by moving Sedge-wick Rafter plankton counting cell from one end to another within focus area. Phytoplankton and zooplanktons/1.0 mL were recorded separately in papers and accordingly planktons of 10.0 mL samples were counted,which were then averaged and calculated as number/L. However, water samples having microalgae less than 70 μm, which were passed through plankton net, were collected into a separate glass beaker. Then 2–3 drops of 4% formalin was added into it and kept as such nearly 2 h for suspension of microalgae. Supernatant water was removed slowly from biker and suspended algal sample was further diluted upto 10.0 ml, of which 1.0 ml was counted as the method mentioned previously.

2.4.5.NPP (Net primary productivity)

NPP was measured by light and dark bottle method (Odum, 1971).The bottles were filled with culture water from respective tanks and incubated vertically 25 cm below the water surface in each tank for 5 h(10.30–15.30 h). NPP status was measured from differences in DO values between Light bottle and Initial bottle using following formula:NPP, mg C/h⋅L =[(Light bottle DO-Initial bottle DO/5 h) × (0.375/1.2)] ×1000

All the analyses were done fortnightly in the 1st experiment and monthly in the 2nd experiment.

2.5.Analyses of microbial load

The groups of microbes such as total coliforms, Salmonella and total viable counts were determined. The aerobic plate count and Salmonella count was done by spread plating the water sample following serial dilution as per APHA (2000). In practice, 0.1 mL of water sample was spread plated in duplicate onto tryptic soy agar aseptically following serial dilutions in 0.85% sterile physiological saline to determine total aerobic counts. Plates were incubated for 24 ± 2 h at 35C ± 2C, and the total viable bacterial colonies were enumerated manually.

Salmonella spp. were detected following the procedure as described by Andrews and Hammack (2007). In brief, pre-enrichment was done by mixing 1 mL of water to 9 mL Tetrathionate broth and incubated at 37C for 24–48 h. Post incubation, a loopful inoculum from the enrichment culture was streaked onto the pre-poured brilliant green agar plate supplemented with 0.08 g/L of sulfadiazine. The presence of Salmonella was determined by red to pink or colourless colonies with a red halo or zone in pre-poured brilliant green agar. Salmonella spp. were identified by biochemical tests of the typical colonies from selective solid medium(APHA, 2000).

The coliform count as expressed in MPN/100 mL was performed by most probable numbers (MPN) in a five-by-three MPN configuration(10-, 1-, and 0.1-mL dilutions) as per APHA (2000) and estimated by referring to Macrady probability table (Cheesbrough, 2006). Tubes were incubated for 24 ± 2 h at 35C ± 0.5C. Yellow color change of the media indicated presence of coliforms.

2.6.Fish sampling, calculation and analyses

Fish were sampled fortnightly interval with drag net from each tank and dead fish were removed timely. Weight of individuals was measured on 90th day. Fish survival (%) was calculated using following formula:

Recorded data were analyzed in one way analysis of variance(ANOVA) using SPSS software version 16.0 for Windows. Data were expressed as mean ± standard error (SE). Duncan’s multiple range tests were used as post hoc to determine the significant differences between means. Comparisons were made at 5% probability level (p <0.05).Statistical analyses were also performed using Statistica 8.0 package to obtain simple linear regression used to represent the relationship: i) the phytoplankton density with parameters such as total ammonia nitrogen and phosphate; and ii) the weight gain (%) offish with parameters such as NPP, phytoplankton and zooplankton. The square of correlation coefficient (rvalue) was used to indicate how well data points fit the linear regression line.

3.Results

3.1.Survival offish in different treatments

Survival (%) of respective fish species in three treatments with no or lower sewage concentrations (Freshwater, 25% and 50% sewage concentrations) were significantly (p <0.05) higher than those in 75% sewage concentration (Fig. 1). The rearing water with 75% sewage concentration was found lethal to more than 50% of stocked fish (33%–46% survival in different species). In this experiment (Table 1), BOD value (mean 41.6 mg/L) in 75% sewage concentration was 22.0 mg/L higher than its nearest BOD value (19.6 mg/L) recorded in 50% sewage concentration. Similarly, the value of total ammonia nitrogen (NH–N)in 75% sewage concentration was significantly higher than those values in freshwater, 25% and 50% sewage concentrations (F =96.422, df =23, p <0.05, Table 1). Increasing level of BOD is related to higher value of NH–N as in 75% sewage concentration.

Fig. 1.Survival (%) offish after 30 days of rearing in different treatments of the first experiment.

Table 1 Physico-chemical parameters of culture water in different treatments of the 1st experiment.

3.2.Dose-response of sewage effluent in relation to water productivity and fish growth

3.2.1.Physico-chemical parameters

Results on changes in hydro-biological parameters compared between split doses and single dose of sewage application were presented in Table 2. The mean value of DO showed increasing trend with the number of split doses (4.04 mg/L in C, 5.85 mg/L in T, 6.83 mg/L in Tand 7.1 mg/L in T), with significant differences (

F

=30.518, d

f

=47,

p<

0.05, Table 2). The values of BOD in different treatments (

F

=0.110,d

f

=47,

p >

0.05, Table 2) were less than 19.5 mg/L and differences among the treatments were not significant. Similarly, the values of ammonia nitrogen (NH–N) and phosphate (PO) were less than 1.0 mg/L and 0.2 mg/L respectively and were not lethal to fish species. The range of pH values (6.73–7.0) in all the treatments (Table 2) was also found similar to that of experiment-1 (Table 1). The value of total alkalinity ranged between 130 and 137 mg/L in different treatments(Table 2), which were found to be suitable for fish survival.

Table 2Physico-chemical parameters of culture water in different treatments of the 2nd experiment.

3.2.2.Plankton assessment

The density of phytoplankton population were significantly high (

F

=3.368, d

f

=47,

p <

0.05, Table 2) in the treatments (T, Tand T)receiving split doses of sewage as compared to control treatment applied with single dose. The highest mean of phytoplankton density (99080 numbers/L) was found in Ttanks, but no significant difference with Tand T(

p

=0.430, Table 2). The density of zooplankton was also significantly (

p <

0.05) higher in both Tand Ttreatments than those in control (C) and TBoth Tand Ttreatments were applied with more numbers of split doses of sewage as compared to control (C) and Ttreatments applied with single and limited split doses of sewage(Table 2).

3.2.3.Microbial assessment

The log transformed levels of total viable count, total coliform count and the

Salmonella

count in the control and different treatments were in the range of 3.93 ± 0.05 to 5.61 ± 0.09 Log cfu mL, 2.79 ± 0.89 to 4.70 ±1.01 Log CFU/mL, and

<

2 Log CFU/mL respectively (Table 2).Microbial load of different treatments (T, Tand T) was remarkably less as compared to that of control. However, no significant difference was found in bacterial count among the treatments (

F

=4.066, d

f

=11,

p >

0.05, Table 2).

3.2.4.Nutrients recovery

Mean value of both total ammonia nitrogen (NH–N) and phosphate(PO), were plotted against phytoplankton density for the experimental period of 90 days (Fig. 2a and b). The value of NH–N was found to be correlated with phytoplankton production in T(

r

=0.59), T(

r

=0.65) and Ttanks (

r

=0.65) with increasing order of nutrient concentration (Fig. 2a). Similarly, the value of POwas also found to be correlated with phytoplankton production in T(

r

=0.41), T(

r

=0.41) and Ttanks (

r

=0.64) with increasing order of nutrient concentration (Fig. 2b). However, phytoplankton density in control tanks was initially high, but decreased with time because of depletion of ammonia nitrogen (

r

=0.48) (Fig. 2a).

3.3.Hydro-biological parameters,fish survival and weight gain

Fish growth, in terms of weight gain (%), was correlated with selected hydro-biological parameters such as NPP, phytoplankton and zooplankton (Fig. 3a, b, c). When NPP was plotted against weight gain,positive correlation was obtained across the species (

r

=0.56 in rohu,

r

=0.66 in mrigal and

r

=0.69 in bata) with increasing trend (Fig. 3a).In biological factors (particular fish species prefers to specific planktons including their size, shape and maturity), phytoplankton density exhibited a strong correlation (

r

=0.86) with weight gain of mrigal,compared to poor correlations with those of rohu (

r

=0.13, Fig. 3b) and bata (

r

=0.09, Fig. 3b). On the other hand, correlations between zooplankton and weight gain of both rohu (

r

=0.94) and bata (

r

=0.90) were found strong, compared to that of mrigal (

r

=0.64) (Fig. 3c).Survival of all fish species was recorded ≥75% in the split dose treatment (Table 3) with BOD values ranging 19.1–19.4 mg/L. There was no significant difference in weight gain (%) of

L. rohita

and

L. bata

reared in different tanks (Control, T, Tand T), but variable weight gain (%) of

C. mrigala

was observed in different treatments (

L. rohu

:

F

=0.035, d

f

=11,

p >

0.05;

C. mrigala

:

F

=673, d

f

=11,

p >

0.05;

L. bata

:

F

=0.186, d

f

=11,

p >

0.05; Table 3).

Fig. 2a.Phytoplankton density in relation to ammonium nitrogen of culture water during the experimental period of 90 days.

Fig. 2b.Phytoplankton density in relation to phosphate of culture water during the experimental period of 90 days.

4.Discussion

4.1.Fish survival in relation to BOD level

Addition of sewage into freshwater causes immediate rise in the BOD level of culture water (Bunting et al., 2010; Chattopadhyay, Saha,Ghosh, & Karmakar, 1988). Increase of BOD above 20.0 mg/L results in oxygen stress that causes fish mortality (Chattopadhyay et al., 1988).The BOD level suitable for wastewater fish culture ranges from 10 to 20 mg/L, and more than this level (>20 mg/L) may develop oxygen stress particularly in late nights (Banerjee, Chattopadhyay, & Boyd, 2010;Chattopadhyay et al., 1988). Evidently, in 75% sewage concentration significantly (p <0.05) high level of BOD (41.6 mg/L) caused fish mortality (Table 1, Fig. 1). Proper loading of sewage is prerequisite to maintain congenial environment in the sewage fed system (Chattopadhyay et al., 1988; Dasgupta et al., 2008; Datta et al., 2000; Jana,1998). Primary treated sewage contains less organic load than raw untreated sewage, but more nutrients than secondary and tertiary sewage.However, certain obnoxious gases, un-ionized ammonia and other chemicals present in sewage make it anaerobic to be unsuitable for direct use (Lahiri et al., 2015). Therefore, proper dilution with freshwater as well as dose is essential to make it aerobic to be conducive for fish survival and growth. In the present experiment, sewage has been suitably loaded through dilution with freshwater which prevents sudden rise of BOD in the culture water conducive for fish survival (Table 2).

Fig. 3a.Scatter plots showing the weight gain (%) of a) Rohu, b) Mrigal and c) Bata against NPP.

Fig. 3b.Scatter plots showing the weight gain (%) of a) Rohu, b) Mrigal and c) Bata against phytoplankton density.

Fig. 3c.Scatter plots showing the weight gain (%) of a) Rohu, b) Mrigal and c) Bata against zooplankton density.

4.2.Dose-response of sewage intake on hydro-biological changes

The consistent high values of DO (6.83 mg/L in Tand 7.1 mg/L in T) due to split doses of sewage facilitates NPP values (194.7 mg C/m⋅h in Tand 202.1 mg C/m⋅h in T) suitable for effective pond water productivity (Table 2). And high DO values are attributed to significantly (p <0.05) higher phytoplankton density found in all the treatments as compared to control. Phytoplankton act as producers and enhance DO in the culture water (Boyd, 2012). The lowest value of DO(4.04 mg/L) in C (control) may be due to the lack of sufficient quantity of phytoplankton in water which suffers from depletion of nutrients. Since control treatment was not applied with any further sewage dose,nutrients amount was not replenished to promote phytoplankton population (Table 2). On the other hand, significantly (p <0.05) highest value of DO (7.1 mg/L) was recorded due to the highest density of phytoplankton in T, which was applied with maximum number of split doses of sewage (24 doses). Earlier studies reveal that addition of nutrients from any source improves the fertilization of pond water (Godara, Sihag, & Gupta, 2015a) resulting in enhanced plankton production used as natural fish food that benefits desirable fish growth (Godara,Sihag, & Gupta, 2015b) and reduction in the pathogenic bacterial populations (Godara, Sihag, & Gupta, 2015c). Application of sewage stimulates phytoplankton production due to supply of nitrogen, phosphorus and trace elements in the same manner as inorganic fertilizers (Banerjee et al., 2010; Chakraborty, Biswas, & Jana, 2004; Chattopadhyay et al.,1988; Edwards, 1992; Jana & Datta, 1996; Lahiri et al., 2015; Mancy,Fattal, & Kelada, 2000; Mandal et al., 2018). In the present study, the nutrients depletion was replenished in T, Tand Ttreatments through addition of sewage with split doses, which was lacking in the control treatment. The present study finds that T, T, and Texhibit gradually higher effective values of recorded hydro-biological parameters due to respective 6, 12 and 24 split doses of sewage.

Table 3Initial weight, weight gain and survival offish after 90 days of rearing in different treatments of the 2nd experiment.

The range of pH values (6.73–7.0) in all the treatments was also observed similar to other studies in the sewage fed system (Chattopadhyay et al., 1988; Dasgupta et al., 2008; Jana, 1998). In the present experiment, diurnal variations of water temperature showed the average values to vary from 31.8C- 32.9C, but no significant difference among treatments. Value of ammonia nitrogen ≥1.5 mg/L becomes toxic to cultured fish (Crab, Avnimelech, Defoirdt, Bossier, & Verstraete, 2007).In the present experiment, the maximum value of ammonia nitrogen recorded below 1.0 mg/L seems acceptable for fish culture.

Coliform count is used as an indicator for assessment of faecal contamination (Gronewold, Borsuk, Wolpert, & Reckhow, 2008). The presence of coliform in all the treatments indicates that contamination occurs through faeces. It is a common incidence because sewage has its origin from the domestic source. In sewage water, the total viable count was in the range of 7.8 × 10CFU/mL to 1.6 × 10CFU/mlL and the coliform count was in the range of 28.84 × 10CFU/mL to 30 × 10CFU mlL. The range of different bacterial count in the present study is consistent to other study (Bhowmik et al., 2000). However, bacterial count has reduced to a considerable level after use of sewage in split doses in the culture water. The higher bacterial count in the control treatment, as compared to the treatments (T, T, T), may be due to the influx of 50% sewage water without any split dose.

4.3.Relationship between weight gain and hydro-biological factors

Ttreatment showed maximal weight gain (%) across fish species followed by T, Tand control treatments, which indicates proper loading of sewage water in the split doses that promote consistent plankton population in rearing water. Otterpohl (2008) opined that optimal rate of sewage intake is essential to achieve desirable fish yield;both high and low amount of sewage hampered the fish production(Jana, 1998; Mandal et al., 2015; Mandal et al., 2018). In a composite fish culture system fed with sewage, mrigal exhibited higher growth than rohu (Datta et al., 2000) because of its bottom dwelling habit.However, in another study, rohu exhibited higher growth than mrigal(Dasgupta et al., 2008). In treatments with split doses, rohu showed maximal weight gain (158.6%–165.7%), followed by mrigal (147.3%–157%) and bata (133.9%–143.8%). In the present experiment,hydro-biological parameters seemed to be influenced by the number of split doses of the sewage water (T) as compared to limited split doses(Tand T) or single dose (C) of sewage application. It seems very effective in sewage fed aquaculture for long duration.

5.Conclusion

For successful fish rearing, we suggest the following measures: (i)dilution of sewage effluent with freshwater to an extent that results BOD level

<

19.5 mg/L in rearing water, (ii) proper loading of sewage may be undertaken through application of split doses to maintain BOD level around 19.0 mg/L in rearing water. In the context of freshwater scarcity,application of sewage in aquaculture ensures water conservation, apart from fertilization of rearing water through supply of potential nutrients which are recovered from organic laden sewage. In the present day aquaculture, the cost of supplementary feed is so high that worries fish farmers. In such a situation, utilization of sewage in aquaculture is useful because it enhances plankton density and thereby, provides a huge amount of natural foods for fish along with sustainable water productivity, which ultimately leads to a desirable fish yield.

CRediT authorship contribution statement

Rathindranath Mandal: Supervision, Writing - original draft,Planning, design, supervise of conducting research and writing the papers. Arabinda Das: Conducting research of aquaculture part. Debnarayan Chattopadhyay: Writing - original draft, Planning, design,and writing the papers. Ajmal Hussan: Conducting research and monitoring day to day research findings. Subhendu Adhikari: Analyses of water quality and their findings with research activities. Baidyanath Paul: Conducting nutritional part and growth offish. Farhana Hoque:Formal analysis, Microbial sampling and analyses. Parthapratim Chakrabarti: Formal analysis, Data collection, recording, statistical analyses and writing papers. Bindu R. Pillai: Writing - original draft,Planning, execution, support and writing the paper.

Declaration of Competing Interest

The authors declare that there is no conflicts of interest.