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Bacterial Population of Fish and Their Environment in Ponds Utilized for Natural Purification of Wastewater

Sutapa Sanyal*, Samir Banerjee

Krishnagar Govt College, Post Graduate Department of Zoology, Krishnagar, Nadia, West Bengal, PIN-741101, India

Abstract: Viable threats from waste-fed aquaculture have become a genuine subject of discussion presumably since the initiation of such fishery. An aquaculture-based sewage treatment system, blending fish as biological component was assessed in Bandipur, Titagarh, North 24 Parganas, West Bengal, India. The 5 MLD (million litres per day) system receiving domestic sewage flow comprised of anaerobic ponds (areas-0.7 ha, depths-2.5m), facultative ponds (areas-4.8 ha, depths-1.5m) and maturation ponds (areas-4.8 ha, depths-1m). The observation revealed substantial aptitude of the system for biological sewage treatment, with respect to reduction of ammonia nitrogen, BOD (Biological oxygen demand) and COD (Chemical Oxygen Demand). The TVC (Total Viable Count) and enteric bacterial counts were detected in higher numbers from water and fish. Some of the members customarily recovered in this study like Vibrio were established fish and human pathogens. Counts of faecal coliforms in fish culture ponds were found to outstrip the World Health Organization (WHO) standards of ≤10³ faecal coliforms per 100 ml of water to be used in aquaculture. High concentrations of pathogenic bacteria were reported to be present in fish even at low numbers of indicatory bacteria. Estimated risk of bacteriological infection of fish for most of the times crossed the guide line proposed by international commission on the microbiological specification of foods (1995). Public health risks associated from fish and fishery products of Bandipur sewage fed farm clearly indicate infelicity of wastewater treatment along with inappropriateness of treated waste water to be reused in aquaculture.

Keywords: aquaculture, wastewater, fish, health risks

Received May 27, 2021; Revised July 05, 2021; Accepted July 14, 2021

Citation: Sutapa Sanyal, Samir Banerjee, Bacterial Population of Fish and Their Environment in Ponds Utilized for Natural Purification of Wastewater. Applied Ecology and Environmental Sciences, 9 (7). 649-655. January 2021. doi:10.12691/aees-9-7-4

1. Introduction

Sewage input in aquaculture ponds spelling prosperity of farmers, is actually a veritable threat to the environment. In the majority of cases, the sewages containing unlimited contaminants are applied untreated. This has added pollution to the water and poison to the organisms. In developing countries, excreta-related diseases are very usual, and wastewater being a source of excreted pathogens like bacteria, viruses, protozoa, and the helminths [1] cause diseases in man. Many of the excreta related infections have definite value in wastewater use schemes [2]. Fish in wastewater is at perpetual risk of contamination by toxic chemicals, heavy metals and pathogenic micro organisms. Skin and digestive tract of fish get frequent contamination and later also internal organs and muscles. Therefore, health hazard to fish handlers and consumers from waste fed fisheries is likely to be very high [3] which demands repeated and extensive prophylactic survey of wastewater fishery.

Few restricted information [4,5,6,7,8] is available about the risks connected to waste fed systems. Detail quality characterization of wastewater is required for water conservation through recycling of wastewater in agriculture, aquaponics and aquaculture. Fish as a cheap source of animal protein to the fish eating population of this country deserves assessment of bacteria in their tissues to provide appropriate information regarding the microbial contamination in the biota and extension of pollution in the environment. In addition to, bacteriological estimation from pathological and toxicological points has predominant importance and becomes crucial if the waste fed fishes are to be commercialized for human consumption.

In view of the public health mater from waste fed fishery and lack of information thereof, principle objective of the present study was to assess the eco health of wastewater aquaculture system. Therefore, the present research work evaluated the bacteriological and chemical contamination of sewage fed aquaculture system. Further, fish can be a forerunner to the emergence of disease epizootics. Hence, present study also scrutinized bacteria present in waste fed fish.

2. Materials and Methods

2.1. Study Site

Waste fed aquaculture system of Bandipur, Rahara, North 24 Parganas, West Bengal, India (22°44'N Latitude and 88º24'E Longitude) was considered for this study. Sewage fed ecosystem consist of anaerobic ponds (the retention times at the designﬂow-1day, pond areas-0.7 ha, and depths-2.5m), facultative ponds (FP, the retention times at the designﬂow-4day, pond areas-4.8 ha and depths-1.5m), and maturation ponds (MP, the retention times at the design ﬂow-4 day, pond areas-4.8 ha and depths-1m) [9]. Design capacity of the waste treatment plant is 14.1 ML/d (million litre/ day) with an average flow of 12 ML/d [9]. Presently wastewater ﬂow is around one- third of the design ﬂow [10]. Ponds are interconnected with cemented sluice gates. Raw sewage is entirely of domestic origin, coming from adjacent Titagarh town and parts of Barrackpore, municipal area of North 24 Parganas, West Bengal, India. Pisciculture is done in both facultative and maturation ponds for the reduction of BOD.

Fish ponds are engaged to raise table size Indian major carps (IMC) viz. Labeo rohita, Catla catla and Cirrhinus mrigala and exotic carps (EC) viz. Oreochromis niloticus, Oreochromis mossambicus, Hypophthalmicthys molitrix and to some extent Cyprinus carpio following multiple stocking and multiple harvesting technique. Fish culture operations are being done in 3 phases in a year.

2.2. Collection and Preparation of Water and Fish Samples

Water samples were collected from the inlet point of the pond storing raw sewage, from facultative ponds and also from maturation ponds for microbiological and chemical characteristics of the samples. Each pond was sampled monthly from June 2008 to May 2011. Water samples were collected from sub–surface i.e., 15 to 20 cm below the water surface to avoid surface contamination. All the samples were brought to the laboratory in a thermo insulated container at 4°C and examined immediately on arrival.

Fish samples were caught with a net and were immediately transferred to the laboratory in containers with pond water. Indian Major Carps like Labeo sp., Cirrhinus sp. along with Oreochromis spp. were subjected to bacteriological assay. Ten live fish of all the species were randomly selected in each season from June 2014 to May 2017.

2.3. Analysis of Physicochemical Parameters of Water

Water samples were stored and fixed for estimation of Temperature, pH, Dissolved oxygen (DO), Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Ammonia nitrogen (AN) and Nitrate Nitrogen (NN) by the standard methods [11].

2.4. Preparation of Sample for Bacteriological Analysis

Appropriate sample dilutions of water were made (10⁻¹-10⁻⁵) with sterile physiological saline (0.85% wt/vol sodium chloride, NaCl) in deionized water.

Dissection, weighing, homogenization and serial dilutions of homogenates of muscles and digestive tract contents of fish were done following [12,13]. Time lag from fish collection to the analyses did not exceed 6 hours.

2.5. Analysis of Indicator Bacteria

All samples (Fish and Water) were examined for the presence of the following organisms: total viable counts (TVC) at 37°C [11], total coliforms (TC) [11], faecal coliforms (FC) [11], Escherichia coli (E.coli) [14] and faecal streptococci (FS) [11].

2.6. Analysis of Pathogenic Bacteria

Fish and Water were also evaluated for the presence of pathogenic bacteria like Salmonella spp., Shigella spp and Vibrio spp. Presence / lack of Salmonella spp., Shigella spp. and Vibrio spp. on enrichment media at 37°C for 24 hours followed by a selective media under same incubation condition were used [11].

Speculative Vibrio spp., E.coli, Salmonella spp., and Shigella spp. were isolated in pure culture and identified by cultural and biochemical characters.

Tests were carried out on each isolate following the procedures delineated by Prescott et al., [15] to enable identification to the generic levels (Table 5) with the aid of the Bergey’s Manual of Determinative Bacteriology [16].

2.7. Statistical Analysis

Logarithmic transformation of bacterial counts was used to normalize data before statistical analysis. All of the results were expressed as mean ± standard errors (SE). Single factor analysis of variance (one way ANOVA) was performed to find the differences in the mean counts of each physico chemical (water) and bacteriological parameters among different treatments and fish species. To determine the differences in the bacterial concentrations mean values of various bacteria obtained from fish tissues (muscles and digestive tract contents) as well as from different treatments (water) were subjected to a two way analysis of variance test (two way ANOVA). In each test difference at P<0.05 was considered significant. The data were analyzed in Excel 2003 (Microsoft Seattle, WA, USA) with the add-in software Statcel 2 [17].

3. Results

Achievements of the waste stabilization ponds were evaluated for the degree of elimination of unenviable contaminants and infectious organisms from the raw sewage. Entire study period was divided into three stages pre monsoon (March to May), monsoon (June to October) and post monsoon (November to February).

3.1. Physicochemical Parameters of Water

Generally the range of variation of water temperature was 26°C in post monsoon to 33°C in pre monsoon. Water in the experimental ponds was slightly alkaline (Table 1). The mean pH values recorded for all sampling points were within the WHO pH tolerance limit of between 6.00 and 9.00 for wastewater to be discharged into environment [18]. Lower concentration of dissolved oxygen was observed in studied sewage fed pond (Table 1). The level of BOD removal was 84% at the facultative ponds and reached 87% at maturation ponds (Table 1). The removal efficiency of COD was 72% at facultative ponds and reached 81% at maturation ponds (Table 1). The ANOVA, P<0.05 for ammonia nitrogen showed that there was significant difference in the mean values among the treatments which was an indication of efficient removal of ammonia from source to out let (Table 1). The ANOVA, P>0.05 (Table 1) for nitrate nitrogen of raw sewage and maturation ponds revealed inefficiency of the treatment system. Nitrate is essentially harmless till its level in the pond water should be controlled to avoid eutrophication [19].

Table 1. Mean±SE (standard error) of physicochemical characteristics of the raw sewage and waste stabilization ponds of the Bandipur. Different superscripts in the same row indicate statistically significant differences between means at P<0.05 (n=12)

3.2. Bacteriological Parameter of Water

Present research found a marked reduction of TVC, TC, FC, E.coli and FS counts as the effluent passed through the different stabilization ponds (Table 2). The sharp reduction in the E.coli counts, which is the most widely used indicator of faecal contamination, represented 70% removal efficiency (Table 2). The two way ANOVA, P<0.05 showed that there was significant difference between bacterial concentrations of various treatment systems (Table 2). Besides, the single factor ANOVA, P<0.05 showed significant difference for each individual bacterial parameters from source to maturation ponds (Table 2). This was further an indication of treatment efficiency. Most of the samples examined for TVC and coliforms in the raw sewage did not comply the WHO guideline [20] (less than 105 FC /100ml for wastewater reuse for aquaculture), but that of the final effluents were very close to WHO levels (Table 2). In entrance points, raw sewage samples were contaminated with Vibrio (100% of tested samples), Salmonella (100% of tested samples) and Shigella (20% of the tested samples), whereas, in exit points bacteriological examination proved the presence of Vibrio (54.5% of tested samples) and Salmonella (16% of the tested samples) but almost complete absence of Shigella (Figure 1).

Table 2. Trends of removal of different indicator bacteria at different steps of treatment in Bandipur sewage fed pond. Values are Mean ± SE (standard error) of twenty four times analysis. Different superscripts in the same column indicate statistically significant differences between means at P<0.05

Figure 1. Trends of removal of different pathogenic bacteria (%) at different steps of treatment in Bandipur sewage fed pond

3.3. Indicator Bacteria in Fish

Faecal indicator bacteria like coliforms and streptococci had been isolated frequently from intestinal tract of various fish species (Table 3). These bacteria are not usually associated with the normal gut flora of fish. Present study showed high faecal coliform labels in fish fillets amounting from 10² to 105 FC/100 g in Labeo sp. (2.4±0.74 to 5.97±0.43 log MPN 100g⁻¹, Table 3) and Oreochromis spp. (2.66±0.90 to 5.14±0.32 log MPN 100g⁻¹, Table 3) and 10³ to 10⁵ FC/100 g in Cirrhinus sp. (2.98±0.79 to 5.02±0.66 log MPN 100g⁻¹, Table 3). High numbers of E.coli were obtained from flesh of all the three fish (Table 3). Fish flesh was also noted to be contaminated by sufficient amount of faecal streptococci (Table 3). TVC of the fish flesh was found in the range of 10⁴–10⁵ cfu/g (Table 3) even at very little faecal coliform penetration. This observation seems to follow the report of [10,12].

No statistical significant differences (Single factor anova P>0.05) were established between the values of different indicator bacterial parameters obtained from muscles of tilapia and carp, the pattern seemed to be similar (Figure 2a. and Figure 3a). Same findings were reported by [12,21]. Similar relationship was observed (Single factor anova P>0.05) for all indicator bacterial in digestive tract contents of the three fish species except for faecal streptococci (Figure 2b. and Figure 3b.).

Table 3. Recovery of bacteria (Mean ± SE, n=24) from individual fish organs

Figure 2. Faecal bacterial counts in muscles (a) and digestive tract contents (b) of different fish species (Similar alphabets represent no significant difference within dataset, n= No. of samples)

Figure 3. Total bacterial counts in muscles (a) and digestive tract contents (b) of different fish species (Similar alphabets represent no significant difference within dataset, n= No. of sample)

3.4. Pathogenic Bacteria in Fish

3.4.1. Vibrio spp

Vibrio spp. were detected in flesh and digestive tract contents of all three types of fish (Figure 4 and Figure 5). Prevalence of Vibrio spp. were high in gut of Cirrhinus sp. than Labeo sp. and Oreochromis spp. (Table 4). Simultaneously flesh of Labeo sp. and Cirrhinus sp. was more contaminated by Vibrio spp. than Oreochromis spp. (Table 4).

3.4.2. Salmonella spp. and Shigella spp.

Pathogenic bacteria like Salmonella spp. and Shigella spp. were almost completely absent in pond water and fish samples (Table 4 and Figure 4 and Figure 5).

Figure 4. Seasonal load of pathogenic bacteria (%) in muscles of fish

Figure 5. Seasonal load of pathogenic bacteria (%) in digestive tract contents of fish

Table 4. Frequency of recovery of pathogenic bacteria from fish species

Table 5. Biochemical characteristics of different bacteria from waste fed fish farm

4. Discussions

The ponds being inter connected, the water temperature remained almost same in all ponds under investigation. The fluctuations in temperature did not make any lethal effect on organisms [22] and remained within the normal range of fish growth [23]. Relatively higher values of pH in Bandipur sewage fed ponds might be due to low rate of sewage input and decrease rate of CO₂ production. Fish mortality due to low dissolved oxygen was not observed. During utmost low concentration of dissolved oxygen fish were aptly noticed at surface for oxygenation averting the stressed condition in the deeper water. Maximum limit of BOD and COD of sewage should be 20 mg/l and 250 mg/l for its discharge into rivers [24]. According to these recommendations, present sewage could be easily discharged into the river. The average ammonia content of the studied fish ponds had crossed the maximum limit of ammonia toxicity without any adverse effects on fish behaviour. Presence of a significant high level of nitrate in the present study suggests that nitrification was probably the principal removal mechanism of nitrogen in the studied system. Further, bacterial load in wastewater clearly indicates that treatment minimizes but does not assure the complete eradication of a putative contamination with bacteria.

The occurrence of faecal coliforms in fish could be an impression of pollution of their aquatic environment in addition to their feeding habits. Moreover, there is affirmation that these bacteria can survive and even multiply with in favorable temperature conditions. Water temperature in the studied pond (26°C to 34°C) was close to optimum temperature for many mesophilic bacteria resulted in high resumption of pollution indicator bacteria in intestine of fish. High bacterial load in intestine of fish might be due to high metabolic activity of fish associated with increased feeding rates in the suitable range of water temperature of the studied pond. Similar findings were reported by [25]. Bacterial growth might occur in intestine with the detention of faecal excreta which is further linked to gut motility. High concentration of phagocytic cells in the intestine, which comprises one of the safeguard to restrict foreign organisms from penetrating the fish, might be another cause for high rate recovery of bacteria from intestine.

Many times coliforms and faecal coliforms were not detected in the muscles of the fish, while other bacteria were recovered. The role of faecal coliform bacteria as indicator in this particular situation is therefore questionable. The detection of significant lower concentration of faecal streptococci in digestive tract contents of Oreochromis spp. than Labeo sp. and Cirrhinus sp. indicates that Oreochrmis is less sensitive to the accumulation of this bacterium than carps. When stressful conditions like low dissolved oxygen or high organic matter concentration occurred in the pond, tilapia appeared to be less affected than carp [12]. High rate recovery of Vibrio from gut of Cirrhinus sp is mainly for its detritivorous feeding habit (bottom feeder) for which it consumes free-living and particle bound bacteria (usually associated with detritus) [26]. Reduced infection of Vibrio in muscles of oreochromis than carps indicates sterile and stress resistant nature of the former fish. [27] showed that aqueous extracts from the skin of both the high saline and freshwater tilapia possessed antimicrobial factors that are antagonistic to luminous Vibrio. Frequent availability of Vibrio in wastewater may be due to halophilic nature of Vibrio spp. as well as suitable temperature of the water as the most important factor of Vibrio distribution [28]. Population density of Vibrio related to the faecal pollution level [29]. [30] reported Vibrio sp. as a stressor in fish growth. Furthermore, anaerobic ponds are worthwhile in removing Vibrio, the classic pathogen that exhibits resistance to the otherwise environmental conditions in facultative and maturation ponds [31]. In fact accumulated sludge from the ponds of the Bandipur sewage fed farm has never been cleaned since the plant was established in 1995 [9]. At present anaerobic ponds filled with accumulated sludge are not working properly. Thus, inability of anaerobic ponds in studied waste fed system might be the reason of high prevalence of Vibrio spp. [32] reported fish as possible reservoirs of Vibrio cholerae. Moreover, Vibrio cholerae isolates have the ability to degrade chitin, indicating a commensal relationship between Vibrio cholerae and fish [33]. Evidences were found from the literature correlating the source of cholera diseases with fish [34]. In the present investigation presence of Vibrio spp. in muscles necessarily implies a health risks. This creates the vital need to assess the species level detection of Vibrios as well as cholera causing O1/O139 or non-O1/O139 serogroups. Rare occurrence of Salomonella and Shigella infection owed to either low excreted load or low survival time of them in environment.

5. Conclusions

It is evident from the investigation that the system offers considerable potential for biological sewage treatment, with respect to reduction of ammonia nitrogen, BOD and COD. The TVC and enteric bacterial counts were detected in higher numbers and some of the members recovered in this study like Vibrio are potential fish and human pathogens. High rate recovery of E.coli from the water of the sewage works also imposes threats because of their pathogenic potentiality or toxin producing ability. Coliforms isolated from fish culture pond were an indication of contamination of pond water with faecal materials, which may result in the presence of pathogenic organisms. Prevalence of faecal streptococci in the pond water might have resulted from pollution by poultry and cattles. Pathogenic bacteria like Vibrio spp. were found frequently in pond water. The organism is ubiquitous in aqueous environments; thus its presence does not necessarily imply a health risk, although non-cholera strains may cause illness in man [35]. In fact, wastewater treatment reduced the number of pathogenic microorganisms but did not remove all the pathogens. Persistence of these pathogenic bacteria, even after wastewater treatment, constitutes a potential risk to cause gastrointestinal diseases. High rate recovery of both indicator as well as pathogenic bacteria in fish muscles could indicate a decrease in the immune response of the fishes living in water with severe environmental quality degradation. Presence of human pathogens such as Escherichia, Salmonella and Vibrio in fish tissues is definitely of prime concern especially in countries with less restriction on release of waste into water bodies, and in use of untreated wastewater for aquaculture. Moreover, Escherichia coli is reported to survive in precooked and uncooked fish when kept in ice. The digestive tract of fish in this study showed concentrations of pathogens. Handling and cleaning of such contaminated fish can result in contamination of the hands of farm workers and through them to their family members and others. Further, pathogens reside in digestive tracts can cross-contaminate fish flesh or other edible parts during processings. Quick spoilage of fish after catching might be due to the high density of these bacteria. Presence of faecal coliform may affect humans more than it does aquatic organisms. In India most of the fish consumed is bought directly from the fishermen and do not pass through any health-safety checks. Bacteria contaminated fish examined in this study exceeded the acceptable limit recommended by [36] and indicate human health risk.

Acknowledgements

The authors are thankful to the Head of the Department of Zoology of University of Calcutta who permitted the research work to be conducted in the department. The author is grateful to Prof. Samir Banerjee for his guidance and invaluable constructive criticism during manuscript preparation.

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