1. Introduction

There is growing scientific interest in understanding the physico-chemical and bacteriological profiles of estuaries or brackish water ecosystems of the Niger Delta region of Nigeria ^{[1, 2, 3]}. The intertidal mudflat of brackish water is the habitat of mangrove oysters. Bivalve shellfish feed by filtering large volumes of brackish water and bioaccumulate food particles ^[4] and microbial pathogens from the surrounding environment ^{[5, 6]}. These microorganisms may present a potential health risk when consumed with raw or lightly cooked shellfish. Contaminated shellfish by industrial, sewage and other anthropogenic inputs may be cleansed by depuration as a shellfish food safety strategy ^{[7, 8]}.

However, the physico-chemical parameters of the habitats are also major determinants of the distribution and composition of the bacterial community ^{[9, 10, 11]}. The efficacy of depuration depends on a number of variables such as the initial health status of the shellfish, environmental parameters within the depuration system, the types of pathogens and level of contamination ^{[7, 12]}. Currently, more attention is being given to depuration protocols ^[8] but with little focus on the dynamics of the physico-chemical factors during depuration.

The objective of this study was to determine the physico-chemical parameters and the bacterial profiles of mangrove oysters, brackish and tap water microcosms in a static depuration system and its potential benefits.

2. Materials and Methods

The mangrove oysters and brackish water samples were collected at both low and high tides from the Gbolokiri creek (at Rumuorlumeni) of the New Calabar River (NCR), Nigeria. The estuary consists of inter-connecting creeks that link up the lower reaches of the NCR. All samples were collected and transported to the laboratory and analyzed within 3-4hours.

Mangrove oysters (200) were randomly collected and washed thoroughly under running tap water and then placed in a sterile rectangular plastic container of 50x35x28cm dimension. Oysters (20) were shucked and analysed at zero-hour bacterial load. The remaining oysters (180) were depurated in 15L of clean brackish and tap water microcosms and the water samples changed at intervals of 24-hours following the method of Chinivasagam ^[13] for 4days. The water samples and oysters were subjected to bacteriological and physico-chemical analyses.

Serial dilutions of the water samples were spread-plated in duplicate on surface-dried plate count agar (Scharlau Chemie S.A. Spain) supplemented with 1.0% NaCl ^[14] and incubated at 37°C for 24hours ^[15] to determine aerobic plate counts (APCs). Preparation of serial dilutions was carried out by blending 25g of oyster meat in 225mL 0.1N alkaline peptone water to obtain a (10^-1) homogenate. Serial dilutions were also prepared for oysters, brackish and tap water samples every 24hours and analysed for bacterial profiles. Total viable colonies (30-300) were enumerated as colony forming units (CFUs).

Coliforms including Escherichia coli colonies were determined on MacConkey agar (Oxoid) using the spread plate method and duplicate plates were incubated at 37°C for 18-24hours. Vibrio colonies were determined on surface-dried thiosulphate-citrate-bile-salt-sucrose agar (Lab M Ltd, UK) using spread-plate method and duplicate plates were incubated at 37°C for 18-24hours.

Identification of bacterial isolates was carried out based on their cultural, morphological and biochemical characteristics ^{[16, 17, 18]}.

The brackish water (BW) samples collected at low and high tides were analysed for several physico-chemical parameters including salinity (‰), pH, turbidity and conductivity as described in Horiba U-10 Instruction Manual ^[19]. Dissolved oxygen (DO) and Biochemical Oxygen Demand (BOD) were determined using the Modified Azide or Winkler’s method in American Public Health Association (APHA) ^[20]. The temperature of the water samples was determined by immersion of a hand-held mercury-in-glass thermometer. The brackish and tap water samples used for depuration were subjected to the same physico-chemical analysis.

Data are presented as mean ± standard deviation (SD). The one way analysis of variance (ANOVA) was used to analyse obtained data for significant differences using SPSS Inc., 2007.

3. Results

The changes in salinity and DO of brackish and tap water microcosms during depuration in dry and rainy seasons are presented in Figure 1. There was an initial increase in the salinity of BW followed by decreases after day 1 of depuration in the dry and rainy seasons. Similar trends were observed with the TW sample but with much lower salinity values. The DO values of BW decreased throughout the depuration period as well as those of TW in the rainy and dry seasons respectively.

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Figure 1. Changes in salinity and DO in brackish and tap water microcosms collected seasonally for oyster depuration. Shaded symbol = Rainy season samples; Unshaded symbol = Dry season samples. Values represent the mean of four determinations. Error bars represent standard deviations of four determination

The pH values were relatively stable in both the BW and TW microcosms during depuration (Figure 2). There was some degree of disparity with the conductivity values of BW during depuration; a high peak was observed on day 3 in the dry season as against absence of peak in the rainy season (Figure 2). The conductivity values were higher during the dry season than in the rainy season.

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Figure 2. Changes in pH and conductivity of brackish and tap water microcosms collected seasonally for oyster depuration. Shaded symbol = Rainy season samples; Unshaded symbol = Dry seasons samples Values represent the mean of four determinations. Error bars represent standard deviations of four determinations

The BW temperature was higher during the dry season than during the rainy season but was relatively stable during depuration (Figure 3). Similar findings were observed with the TW (Figure 3). There was a sharp increase in BW turbidity as opposed to TW during depuration in the rainy and dry seasons respectively.

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Figure 3. Changes in temperature and turbidity of brackish and tap water microcosms collected seasonally for oyster depuration. Shaded symbol = Rainy season samples; Unshaded symbol = Dry seasons samples. Values represent the mean of four determinations. Error bars represent the standard deviations of four determinations

There were initial sharp decreases in BOD values for both samples and thereafter followed by progressive increases after day 1 for the BW and decreases for the TW in BOD values for both seasons respectively (Figure 4).

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Figure 4. Changes in BOD of brackish and tap water microcosms collected seasonally for oyster depuration. Shaded symbol = Rainy season samples; Unshaded symbol = Dry seasion samples. Values represent the mean of four determinations. Error bars represent standard deviations of four determinations

In general, there were decreases in bacterial loads between days 0 and 1 followed by progressive increases except for E. coli (Table 1 and Table 2). Evidently, the APCs, CCs and VCs were higher for the dry season than those for the rainy season for both BW and TW. Comparatively, the APCs of oysters depurated in TW microcosm were higher than those of BW irrespective of seasons.

Table 1. Bacterial population (cfu/g) dynamics of oysters depurated in brackish and tap water microcosms during rainy season

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Table 2. Bacterial population (cfu/g) dynamics of oysters depurated in brackish and tap water microcosms during dry season

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The bacteria isolated from the samples are shown in Table 3. The most diverse bacterial genera occurred in the brackish water while the least diverse was observed in the tap water microcosms.

Table 3. Bacterial flora isolated from tap water, oyster and brackish water samples during depuration

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4. Discussion

The results of this investigation indicate that appreciable changes occurred in the physico-chemical parameters, bacterial profiles of the microcosms and the oyster samples (Figure 1-Figure 4 and Table 1-Table 3). The progressive decreases in salinity during depuration of oysters in both microenvironments may be related to the ability of bivalves to assimilate substances from their environment, digest and discharge some in form of pseudofaeces ^{[21, 22]}. The decreases in DO in both microcosms during depuration may not be unconnected with the presence of materials of high organic content leading to oxygen depletion (Figure 1). The occurrence of DO deficit is indicative of deoxygenation rate due to higher biological decomposition of organic matter compared with reoxygenation from the microenvironment or may be due to the presence of oxidizable minerals in the microcosms of stagnant water which has been reported by previous workers ^{[23, 24, 25]}. The DO of not less than 2.0mg/l has been recommended ^[22] as conducive for depuration of bivalve. Thus, the DO values observed in the depuration microcosms were adequate for the oysters.

The relatively stable pH values of both microenvironments may be attributed to the activities of microorganisms which make their environment more alkaline by generating ammonia through amino acid degradation or production of acidic or basic metabolic waste products ^{[7, 26, 27]}.

The increase or fluctuation in conductivity values of the brackish water microenvironment may be due to the concentration of ions by evaporation, coupled with increased mineralization of organic matter in the stagnant unagitated water. On the other hand, the relatively stable conductivity values of TW microenvironment could be attributed to poor conductivity and ionic concentration while those of the BW reflect the status of inorganic constituents ^[25]. The concentrations of ions of metals and nonmetals have been earlier reported to affect the metabolism of bacteria and higher organisms in brackish water ^[28]. This may partly explain the shorter shelf-life of oysters in TW than in BW during the depuration.

Similarly, the consistent temperatures observed throughout depuration suggest their probable beneficial effects on the ecology of oysters and their sustainability. But the progressive increases in turbidity of both water bodies may not be unrelated to the metabolic activity of organisms and the discharge of pseudofaeces in the microenvironment. In addition, the natural status of the water used for depuration may contribute to changes in turbidity. Lio-Po ^[22] had earlier reported 77NTU as the maximum allowable turbidity in a depuration system and the results obtained in this work are lower and hence adequate for the physiological activities of the oysters.

The observed increases in BOD (Figure 4) in the BW microenvironment could be related to rapid biological depletion of organic matter as a result of proliferation of diverse bacterial groups (Table 3). In contrast, the decrease in BOD of TW microcosm may not be unconnected with the composition of the medium and the bacterial content. Consequently, the poor activity of oysters in TW may be related to the influence of physico-chemical parameters and these may explain the higher bacterial loads in TW than in BW microcosms (Table 1 and Table 2).

Depuration has been one the strategies used in eliminating oyster-associated bioindicators, microbial pathogens and viruses ^{[6, 8, 28]} and therefore constitutes a veritable tool for assessing and achieving microbial safety of oysters. The APCs in oysters depurated in BW and TW microcosms during the dry season were comparatively higher than in the rainy season (Table 1 and Table 2) most probably due to evaporation effects. The apparent decreases in APCs following day 1 of depuration were indicative of the beneficial effects associated with purification of contaminated shellfish ^{[7, 27, 29]}. The increases in APCs after day 1 may be attributed to selective mechanism which operates in the gut and intervalvular fluid of oysters that favours the retention and multiplication of some microorganisms such as gram-positive cocci and Vibrio species ^{[30, 31]}.

The present work has also shown that coliforms, especially E. coli were not selectively retained in the gut but were readily eliminated from the oyster during depuration such that E. coli became undetectable after day 1 (Table 1 and Table 2). This corroborates the findings earlier reported by other workers ^{[31, 32]}. The reduction in APCs and CCs in BW microcosms than in TW used for oyster depurations in both rainy and dry seasons (Table 1 and Table 2) further confirms the beneficial impacts of BW in terms of enhanced microbial safety.

5. Conclusions

The physico-chemical parameters of the microcosms influenced the diversity and composition of bacterial flora as well as the activity and viability of the oysters. However, this study has provided baseline information on the range of physico-chemical parameters needed to understand the dynamics of bacterial communities available in the depuration system. It also showed that the conditions affecting oyster activity during depuration in the microcosms drastically affect the profiles and manner of bacterial elimination.

References

[1]	Amadi, L.O. and Efiuvwevwere, B.J.O. Seasonal variations in physico-chemical and bacteriological parameters of Ogodogbo creek, Niger Delta Nigeria. International Journal of Environment and Bioenergy, 2015, 10(2):94-106.
	In article
[2]	Edun, O. M. and Efiuvwevwere, B.J.O. Bacterial profiles and physico-chemical parameters of water samples from different sites of the New Calabar River, Nigeria. Sustainable Agriculture Research, 2012, 1(2):162-169.
	In article		View Article
[3]	Odokuma, L.O. and Ijeoma, S.O. Seasonal changes in the heavy metal resistant Bacterial population of the New Calabar river, Nigeria. Global J. Pure and Applied Sciences, 2003, 9 (4): 425-433.
	In article
[4]	Iwamoto, M., Ayers, T., Mahon, B.E. and Swerdlow, D.L. Epidemiology of Seafood-Associated Infections in the United States. Clin. Microbiology Reviews, 2010, 23(2):399-411.
	In article		View Article  PubMed
[5]	Hatha, A.A.M., Christi, K.C., Singh, R. and Kumar, S. Bacteriology of the fresh water bivalve clam Batissa violacea (Kai) sold in the Suva market. The South Pacific J. Nat. Sci. 2005, 23: 48-50.
	In article
[6]	Lee, R., Younger, A. and Dorẻ, B. Emerging Issues in Water and Infectious Disease Series- Safe Management of Shellfish and Harvest Waters. Eds. G. Rees, 2010.
	In article
[7]	Obodai, E.A., Nyarko, H.D. and Amponsah, S.L. Effect of depuration on microbial content of mangrove oyster (Crassostrea tulipa, L) from Benya Lagoon, Ghana. Ethiopian Journal of Environmental Studies and Management, 2010, 3(2): 47-53.
	In article		View Article
[8]	Scottish Aquaculture Research Forum (SARF). SARF066/FD3.0 Review and cost-benefit analysis for industry of reduced depuration times for the mussel Mytilus edulis. Published by SARF, UK. https://www.sarf.org.uk, 2011.
	In article
[9]	Yannarell, A.C. and Triplett, E.W. Within and between lake variability in the composition of bacterioplankton communities: Investigations using multiple spatial scales. Applied Environ Microbiol., 2004, 70:214-223.
	In article		View Article  PubMed
[10]	Hahn, M.W. The microbial diversity of inland water. Curr. Opin. Biotechnology, 2006, 17: 256-261.
	In article		View Article  PubMed
[11]	Wu, Q. L. and Hahn, M.W. Differences in structure and dynamics of Polynucleobacter communities in a temperate and a subtropical lake revealed at three phylogenetic levels. FEMS Microbiol. Ecol., 2006, 57:67-79.
	In article		View Article  PubMed
[12]	Jackson, K.L. and Ogburn, D.M. Review of depuration and its Role in Shellfish Quality and Assurance. Final report to Fisheries Research and Development Corporation- Project No. 96/355, 1999.
	In article
[13]	Chinivasagam, H.N. Status of Sanitation and Marketing of Mollusc in Sri Lanka. In: FAO Corporate Document Repository. Report of the workshop and study tour on mollusc sanitation and marketing. Regional seafarming development and demonstration project RAS/86/024. France, 1989.
	In article
[14]	Dalgaard, P. Freshness, Quality and Safety in Seafoods. FLAIR-FLOW Europe Technical Manual. F-FE 380A/00. EU- funded projects, 2000.
	In article
[15]	Anon. Standard Methods for the Examination of Water and Wastewater, 16th ed. Washington, DC, U.S.A, 1985.
	In article
[16]	Cowan, S.T. and Steel, K.J. Manual for the identification of medical bacteria 2nd edn. Cambridge University Press, Cambridge, 1974.
	In article
[17]	Harrigan, W.F. and McCance, M.F. Laboratory Methods in Food and Dairy Microbiology. Academic Press, London, UK, 1976.
	In article
[18]	Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G. Bergey’s Manual of Systemic Bacteriology,Vol 2, Williams and Wilkins, Baltimore, MD, USA, 1986.
	In article
[19]	Horiba Instruction manual. Water Quality checker U-10 2nd edn. Tokyo, Japan, 1991, pp. 54-67.
	In article
[20]	American Public Health Association Standard methods for the examination of water and wastewater 18th edn. American Public Health Association, Washington, DC. U.S.A, 1992.
	In article
[21]	Quayle, D.B., Tropical Oysters: Culture and Methods. Ottawa, Ontario, IDRB-TSITE, 1980, p 80.
	In article
[22]	Lio-Po, G. Post-Harvest Technology of Commercially Important Bivalves. Selected papers on mollusk culture. In: FAO Document Repository. FAO Regional seafarming Development and Demonstration Project (RAS/90/002), 1990.
	In article
[23]	Das, J. and Acharya, B.C. Hydrology and assessment of lotic water quality in Cuttack City, India. Water, Air and Soil Pollution, 2003, 150:163-175.
	In article		View Article
[24]	Gasim, M.B., Ismail, B.S., Toriman, E., Mir, S.I. and T. C. Chek. A Physico-chemical Assessment of the Bebar River, Pahang, Malaysia. Global Journal of Environmental Research, 2007, 1(1):7-11.
	In article
[25]	Tukura, B. W., Gimba, C. E. Ndukwe, I. G. and Kim, B. C. Physico-chemical characteristics of water and sediment in Mada River, Nasarawa state, Nigeria. Int. J. Environ. Bioenergy, 2012, 1(3): 170-178.
	In article
[26]	Prescott, L.M., Harley, J.P. and Klein, D.A. Microbiology, 3rd edn. WCB Wm. C. Brown Publishers, U.S.A, 1996, pp. 880-885.
	In article
[27]	Efiuvwevwere, B.J.O. and Ezeama, C.F. The bacteriological profiles of freshwater snail (Pila ovata) subjected to microcosms simulating local storage conditions. World Journal of Microbiology and Biotechnology,2004, 20: 359-363.
	In article		View Article
[28]	Gill, O.N., Cubitt, W.D., McSwiggan, D.A., Watney, B.M. and Bartlett, C.L. Epidemic of gastroenteritis caused by oyster contaminated with small round Structured viruses. Br Med J (Clin Res Ed), 1983, 287(6404):1532-1534.
	In article		View Article
[29]	Murphree, R.I. and Tamplin, M. L. Uptake and retention of Vibrio chlorae 01 in the Eastern Oyster, Crassostrea virginica. Applied and Environmental Microbiology, 1995, 61, 3656-3660.
	In article		PubMed
[30]	Vasconcelos, G. J. and Lee, J.S. Microbial Flora of Pacific Oysters (Crasssostrea gigas) subjected to Ultraviolet-Irradiated Seawater. Applied Environmental Microbiology, 1972, 23(1):11-16.
	In article
[31]	Maalouf, H., Zakhour, M., Le Pendu, J., Le Saux, J-C. Almar, R. and Le Guyader, F.S. Distribution in Tissue and Seasonal variation of Norovirus Genogroup I and II Ligands in Oysters. Applied and Environmental Microbiology, 2010, 76(16):5621-5630.
	In article		View Article  PubMed
[32]	Souness, R.A. and Fleet, G.H. Depuration of the Sydney rock oyster (Crassostrea commercialis). Food Technology Australia, 1979, 31:397-404.
	In article