CHAPTER ONE

INTRODUCTION

1.1 Background of the study

Fish farming has important role in meeting the increase demand for fish production. Poor

water quality of pond acts as predisposing factors for many infectious diseases which can

cause mass mortality in freshwater fishes (Krupa, Prasanna, Prejit, Anoopraj, and Ajith,

2019). Fish and its product are very important to human population all over the world (Njoku,

Agwa, and Ibiene, 2015). This is because it is the preferred source of animal protein as

compared to others (i.e. poultry, beef, mutton or pork) (Delgado, Parisi, and Vaz-Almeida,

2017). It is comparatively cheaper and highly acceptable with little or no religious preference

which gives it advantage over other proteins, benefiting the body with high biological value

in terms of high protein retention in the body, low cholesterol level and presence of essential

amino acids (Adewuyi et al., 2010; Emikpe et al., 2011). Fishery products are not only

important from a nutritional point of view, but also important for international trade, foreign

exchange for a number of countries in the world and creation of job opportunities (Adebayo

et al., 2012; Béné et al., 2016).

Agriculture is an integral part of the Nigerian economy employing over 70% of the active

labour force. Catfish production plays a major role in Nigeria aquaculture industry given that

it is the largest segment of aquaculture in Nigeria (Oluwatayo and Adedeji, 2019; Garlock et

al., 2020). Most catfish are cultured in the southern part of Nigeria, and the industry is

economically important to several other states (Omofunmi et al, 2017).

Fishes are cultivated in different culture media or controlled environment which could be

ponds (concrete or earthen), vats (wooden or fiber glass) and plastics. The most widely used

cultivation medium is the concrete and earthen ponds (Fakorede et al., 2020). Earthen pond
culture system has been the traditional method of fish cultivation in Nigeria, until recently the

concrete pond culture system is in use as land becomes scarce, expensive and unavailable. In

Nigeria it has been estimated that 73% of fish farmers make use of concrete ponds compared

to the 27% using earthen ponds. The water quality of a fish farm is determined by its

physicochemical and biological properties (Famoofo et al., 2020). The determinants of the

water quality may include the questionable sources of water, the feed used for the fish which

are produced from animal manure which may serve as a suitable substrate for a wide variety

of microbial growth in the pond. Water sources for earthen ponds are usually untreated

surface water runoffs from streams, rivers, lakes, stored waters while underground water

source is being utilized for most concrete ponds. Water from underground sources such as a

well is pumped and stored in storage tank which is used to refill the ponds when the water is

to be changed at intervals of about 3-5 days (Kim et al., 2021). The feed used for fish in these

ponds contain organic materials from cow dungs and introduces a wide variety of

microorganisms into the ponds. Bacterial pathogens isolated from the fish feed include: E.

coli, E. coli 0157:H7, Shigella dysenteriae, Staphylococcus aureus, Salmonella typhi, Vibrio

cholerae, Klebsiella pneumoniae and Aeromonas hydrophila (Omojowo and Omojasola,

2013; Ramírez-Castillo et al., 2015). The microbial flora of a cultivated fish is a reflection of

its aqueous environment (Njoku et al., 2015).

Wastewater comprises liquid waste containing a wide range of potential contaminants.

Wastewater contains offensive and possibly dangerous substances which are mostly of

anthropogenic origin and causing pollution and contamination in the surrounding

environment. Discharge of untreated wastewater pollutes the soil and surface water and this

could be worsened during flooding (Edokpayi et al., 2017). Heavy metals such as cadmium,

zinc, mercury, chromium, copper, cobalt, nickel, manganese, iron, vanadium and

molybdenum from industries cause heavy pollution particularly in the ponds, lakes and river
systems increasing the Biological Oxygen Demand (BOD). The use of indicator bacteria such

as faecal coliforms in water quality determination is widely used. Coliforms and Escherichia

coli are of great importance among bacterial indicators used in water quality assessment.

Physico-chemical parameters such as dissolved oxygen, pH, temperature, conductivity, total

alkalinity, total hardness, total solids, transparency values, carbon dioxide, nitrite-nitrogen,

sulphates, carbonates and ammonia are some of the salient factors to consider in relation to

fish health (Fafioye, 2011).

1.2 Justification of the study

The high demand of fish has resulted to the increase in the number of fish pond in Calabar

metropolis. Individual farmers, organized groups and institutions have developed and

construct fish ponds and started fish farming oblivious of the cost. The wastewater from these

fish ponds are often discharged into the surrounding drains. Thereby introducing some

pathogenic organisms to the surrounding water bodies and farmlands. Hence, this study was

conducted to determine the quality of the wastewater in order to subject such water to proper

treatment to protect the environment, plant, animal and man. This study will throw more light

on the chain of transmission of infection and necessary management systems required for

microbiological quality maintenance of pond water sources. The study will also help policy

makers to guide informed decision making in respect to wastewater treatment and discharge

process and serve as a link for further research.

1.3 Aim and objectives

The aim of this study is to carry out bacteriological examination of fish pond effluents from

selected fish pond in Calabar Metropolis.

1.3.1. Specific objectives

The specific objectives of the study are to;

 1. Determine total heterotrophic count, total coliform and faecal coliform from the

drinking water samples.

2. Identify and characterize bacterial isolates from the sampled fish pond effluents in the

study area

3. Determine the prevalence of occurrence of bacterial isolates from selected fish ponds

in the study

1.4. Operational Definition of Terms

Aquaculture: This is also known as aquafarming; it is the controlled growth of aquatic

species.

Fish Farming: This is the form of aquaculture in which fish are reared in enclosures to be

sold as food.

Fish pond: This is a controlled pond, small artificial lake or retention basin that is stocked

with fish and is used in aquaculture for fish farming for recreational fishing, or for

ornamental purposes.

Wastewater: These are water generated after the use of fresh water, raw water, drinking

water or saline water in a variety of deliberate applications or processes.

 CHAPTER TWO

LITERATURE REVIEW

2.1 Overview of Aquacultural Practices

Aquaculture is one of the world's fastest increasing industries of agriculture (FAO 2014).

Aquaculture is a significant financial activity in many nations and provides possibilities for

poverty alleviation, jobs, community development, decrease of natural resource exploitation

and food security in tropical and subtropical areas. Aquaculture can also have immediate

adverse effects on wild fish, birds and mammals such as seals and sea lions. Aquaculture, the

fastest increasing significant food sector for centuries, presently generates 53% of the

worldwide fish we eat and is mainly liable for increasing the consumption per capital of fish

goods since the 1960s (SOFIA, 2018). Global fish production peaked at approximately 171

million tons in 2016, with aquaculture accounting for 47% of the total and 53% excluding

non-food uses (including reductions in fishmeal and fish oil). Fish and fish products are today

some of the world's most widely traded food items.

In 2016, for human consumption or non-edible purposes, around 35 percent of worldwide

fish manufacturing joined international trade in multiple forms. The 60 million tons of total

fish and fish products exported in 2016 (live weight equivalent) constitute a rise of 245

percent over 1976. World trade in fish and fish goods also increased considerably in terms of

value during the same era, with exports increasing from USD 8 billion in 1976 to USD 143

billion in 2016.

Despite significant modifications in absolute manufacturing, the prevalent uneven pattern of

distribution of manufacturing between areas and nations within the same area has stayed

pronounced and mainly unchanged over the previous century. For more than two decades,

Asia has accounted for around 89 percent of world aquaculture manufacturing. Africa and the

Americas have raised their corresponding shares in complete world manufacturing over the
same period, while Europe's and Oceania's shares have significantly fallen. Over the previous

two decades, Egypt, Nigeria, Chile, India, Indonesia, Viet Nam, Bangladesh and Norway

have reinforced to differing degrees their share of regional or world manufacturing. With

greater population growth and growing economically active populations in the agricultural

sector, Africa and Asia have shown a usually favorable trend in the amount of individuals

involved in fishing and greater rates of development in those involved in aquaculture.

Without referring to Nigeria, the increase in African contribution to world fish production

and the rapid growth of aquaculture in Africa cannot be discussed. Being the second highest

producer of cultured fish in Africa (second to Egypt) and the highest producer of the second

most important aquaculture product in Africa (Clarias gariepinus) (FAO, 2012), Nigeria has

witnessed a rapid development in aquaculture production in recent time, considering the

production level of about 40 000 metric tonnes (only 6% of the total domestic fish

production) in 2006 to 200 535 metric tonnes (24.4% of the total domestic fish production) at

the end of year 2010 (Adeogun et al., 2017)

2.2 Economic Importance of Fish

People in the world depend on fish as a supplement of their food nutrients. Fish has become

an increasingly important source of protein and other elements necessary for the maintenance

of a healthy body (Emikpe et al., 2011). Fish and seafood constitute an important food

component for a large section of the world’s population. Throughout history; humans have

used fish as a source of protein, Fish is the third staple animal protein and forms a cheap

source of protein. Fish and fish products constitute an important part in the international

trade; more than 50 billion fish are eaten annually indicating increasing consumer interests in

the commodity (Wafaa et al., 2011). In Africa, as much as 5 % of the population depends

partly or wholly on fish for their livelihood. Aquaculture has become one of the fastest
developing source of animal protein to humans and animals due to dwindling wild fish stocks

around the world and in particular Ghana. In Nigeria, the rearing of African cat fish is

proving to be a lucrative option for small scale inland fisheries.

Unfortunately, most of these are obtained through naturally occurring fish from the wild and

partly through fish importation. Generally, fish are good sources of vitamins B12 and B6, it is

also a good source of fluorine and iodine which are needed for development of strong teeth

and the prevention of goiter in man (Adebayo-Tayo et al., 2012). Economic studies have

demonstrated that fish farming in Africa can be a good source of income. Fishery products

are important not only from a nutritional point of view, but also as an item of international

trade and foreign exchange earner for a number of countries in the world. Nigeria is the

largest African aquaculture producer with over 600,000 metric tonnes a year. Egypt with over

400,000 metric tonnes follows Nigeria, and then there are only five other countries (Zambia,

Madagascar, Togo, Kenya and Sudan) that each produce more than 100,000 metric tonnes

(Egbere et al., 2018).

2.4 Types of Culture Structure

Aquaculture has been practiced successfully in different holding structures ranging from

ponds, tanks, raceways, cages and pens.

Ponds: A pond is a controlled pond, artificial lake, or reservoir that is stocked with fish and

is used in aquaculture for fish farming, or is used for recreational fishing or for ornamental

purposes. Mostly earthen ponds are used for culture of carps, tilapia, catfishes and sea bass

(Ozigbo, 2014).

Auxiliary Ponds: These include ponds for segregation of food stock, spawning ponds, Fry

nursing ponds, Fry holding ponds, Storage ponds for marketable fish, Overwintering ponds.

They usually much smaller ponds and may serve different functions in different seasons as,
the same pond can be exploited for carp spawning in the spring, fry nursing in the summer,

fish retention in the fall, and fry overwintering in the winter. Often, 85% of the farm's total

pond area is made up of growing ponds, while 15% is made up of auxiliary ponds (Magdy,

2016).

Raceways: One of the pioneering techniques for inland aquaculture is raceway systems. A

raceway generally contains either concrete canals or basins that are rectangular in shape and

have an inlet and outflow. A continuous water flow through is maintained to provide the

required level of water quality, which allows animals to be cultured at higher densities within

the raceway. It usually comprises a parallel sets of a narrow channels constructed in

sequential blocks with two to three raceways sets in series. Typically, it is about 30×3×1 m

they may be smaller or larger having fast water flow rates (Magdy, 2016).

Tanks: However, fish farming can be done in concrete or plastic tanks that may be indoor or

outdoor. Small tanks (made of glass or plastic) or huge fiberglass tanks can be utilized as

tanks. Although production tanks come in a variety of sizes and shapes, round tanks with

capacities of 5,000 to 10,000 liters are the most widely used (ALSS, 2013). Tanks must be

non-corrosive; hence fiberglass or plastic are recommended materials. Conical-shaped

bottoms on smooth circular tanks are thought to be useful because they help with the release

of waste materials during draining. Water enters the tank through pumps in a way that causes

a circular circulation within, and it exits through the center of the tank through a drain pipe or

a bottom drain that is encircled by screen. Draining out is designed to occur through a central

outlet made up of a drain pipe that is enclosed by screen. Security is much easier with a tank

system because production is concentrated on a small site, which can completely fence in

(Magdy, 2016).

Floating Cages: This refers to the rearing of fish animals in floating cages with a net

suspended below, within barriers in natural waterways. They could be circular, square, or
rectangular. To keep fish confined and safe until they can be harvested, fish cages are

installed in lakes, bayous, ponds, rivers, and oceans. When the cages are submerged beneath

the water, the method is also referred to as "off-shore cultivation." When they reach market

size, they are taken 9 after being artificially fed and maintained in cages. Several ecosystems,

including rivers and streams, saltwater estuaries, and coastal marine zones, are implementing

open systems. The size of floating mesh cages, which are anchored to the ocean floor, varies

depending on operation's scope and the species cultured (Ozigbo, 2014).

Pens and Net Enclosures: In shallow water, usually in ponds, pens and net enclosures

(hapas) are used to establish a restricted environment for the culture of fish and some

crustaceans. They are usually not really big, measuring only a few tens of square meters or

fewer. Tightly packed stakes, such as bamboo stems or mangrove branches, or wire and other

mesh can serve as the enclosures' walls. M most of these cultures are practiced in

undeveloped countries. The containment of bottom-dwelling scallops with mesh fences or

walls is an interesting exception to the shallow water pen. These pens are high enough to

keep the scallops from swimming over the wall. They could use floats to allow the mesh to

move with the tide. Haps are net enclosures made of fine nylon, plastic mosquito netting, or

cotton mesh. Harvesting is much easier with haps because the fry cannot escape. Haps can be

any manageable size ranging from 140 m3 to 1-2 m in depth and suspended on poles. Simple

squares or rectangles can be used. There are also more complex designs, such as those that

use a series of nets to separate brood stock from fry.

2.4.1 Sources of Waste from Aquaculture

(a) Feed

Depending on the culture technique utilized, feed plays a different role in aquaculture

productivity. The principal source of waste in aquaculture systems has been recognized as

feed (Martins et al., 2010; Akinwole et al, 2016). The amount of extra feed has an impact on
the amount of fish feed waste produced. Nutrient content, manufacturing method (extruded

vs. pelleted), feed size to fish size ratio, quantity of feed per unit time, feeding method, and

storage period are just a few of the variables that affect waste formation from feed (Miller &

Semmens,2012).

(b) Chemicals

Chemical use in fish farms is strictly limited in current aquaculture practices; however, some

chemicals are still used in the form of medications, disinfectants, and antifoulants. Antibiotics

are used for prophylaxis and curative purposes are anesthetics, ectoparasiticides,

endoparasiticides, and vaccines, which are used for the treatment and control of parasites

(internal and external), as well as microbial infections (Dauda et al., 2018). Salts are

primarily used to reduce stress in fish, while lime is used to treat pond bottoms for acidity

during pond preparation, and other chemicals that are not harmful to fish are also used.

Although these chemicals are beneficial to fish culture, they may also be harmful to humans.

As the water is released from the ponds, it flows into natural water bodies. The effect of these

chemical wastes upon these natural water systems depends on the concentration of chemicals

used, the farm size, and the size of the receiving water bodies.

(c) Pathogens

This group of waste is rarely considered in aquaculture systems, especially when it is below

the level that affects the cultured fish. However, discharging pathogens with the wastewater

may negatively affect the aquatic organisms in the natural water bodies. Natural water bodies

have their own pathogenic load and receiving additional loads from fish culture systems may

cause stress or the outright death of aquatic organisms. The discharge of pond effluent is

rampant in semi-intensive pond aquaculture, which is more common in Africa, where organic

fertilizers used in aquaculture resulted in a high level of pathogens. Four organic fertilizers
(blood cow waste, cow manure, pig manure, and poultry manure) contribute to a high level of

fecal streptococci (Ampofo and Clerk, 2003).

2.4.2 Characteristics of Wastewater

2.4.2.1 Physicochemical Characteristics

The physicochemical characteristics of wastewater that are of special concern are pH,

dissolved oxygen (DO), oxygen demand (chemical and biological), solids (suspended and

dissolved), nitrogen (nitrite, nitrate and ammonia), phosphate, and metals. The hydrogen-ion

concentration is an important quality parameter of both natural and waste waters. It is used to

describe the acid or base properties of wastewater. A pH less than 7 in wastewater influent is

an indication of septic conditions while values less than 5 and greater than 10 indicate the

presence of industrial wastes and non-compatibility with biological operations. The pH

concentration range for the existence of biological life is quite narrow (typically 6-9). An

indication of extreme pH is known to damage biological processes in biological treatment

units (Akpor & Muchie, 2011).

Unlike organic pollutants, they cannot be degraded, but accumulate throughout the food

chain, producing potential human health risks and ecological disturbances. Their presence in

wastewater is due to discharges from residential dwellings, groundwater infiltration, and

industrial discharges. The accumulation of these metals in wastewater depends on many local

factors, such as the type of industries in the region, way of life and awareness of the impact

on the environment through the careless disposal of wastes (Hussein et al., 2015; Silvia et al.,

2016).

2.4.2.2 Microbiological Characteristics

Municipal wastewater contains the most common types of microorganisms, including viruses,

bacteria, fungus, protozoa, and helminthes. Although different aquatic microbes are thought
to have a significant part in a number of waterborne epidemics, including pneumonia,

diarrhea, meningitis, degenerative heart disease, and stomach ulcers, they also have a number

of positive effects on wastewater influents (Kris, 2017). In secondary wastewater treatment,

dissolved organic matter is often removed using microorganisms. The microorganisms are

utilized in fixed film systems, suspended film systems, or lagoon systems depending on the

treatment plant's preference. Their presence can promote solids breakdown, resulting in

decreased sludge generation, during the various treatment phases. Aside from solid reduction,

wastewater microbes also recycle nutrients such as phosphate, nitrogen, and heavy metals. If

nutrients trapped in dead materials are not broken down by microbes, they will never be

available to help other organisms survive. Microorganisms are also in charge of detoxifying

acid mine drainage and other toxins in wastewater. Microbial pollutants can also be used as

water quality indicators. Detecting, isolating, and identifying various types of microbial

pollutants in wastewater is always difficult, expensive, and time consuming. In order to avoid

this, indicator organisms are always used to assess the relative risk of the presence of a

specific pathogen in wastewater (Paillard et al., 2005).

For instance, enteric bacteria, such as coliforms, Escherichia coli, and faecal Streptococci are

used as indicators of faecal contamination in water. To indicate viral contamination,

bacteriophages (somatic and F-RNA coliphages) are used. Also, Clostridium perfringens, a

faecal spore-forming bacterium, which is known to live longer in the environment and

reported to be resistant to chlorine, is used as an indicator for the presence of viruses,

protozoa or even helminthes eggs. Furthermore, diatoms are used to indicate the general

quality of water with respect to nutrient enrichment, and they provide valuable interpretations

with respect to changes in water quality, such as turbidity, conductivity, COD, BOD and

chloride (Dela et al., 2012).

2.5 Components of Wastes from Aquaculture Systems

Generally, wastes from aquaculture can be classified into solid wastes, dissolved wastes and

microbial waste.

(a) Solid Wastes

Solid wastes are primarily derived from the uneaten feed and fecal droppings of cultured fish.

They occasionally include those fish that do not survive the culture process. Solid wastes can

be further classified as suspended solids and settled solids. The suspended solids are fine

particles and remained suspended in the water, except when a method of coagulation or

sedimentation is employed, and are the most difficult type of solids to remove from culture.

Solid wastes have been classified as the most dangerous waste in fish culture systems and

should be effectively removed as quickly as possible. Solid wastes are regarded to be very

dangerous because they can clog the fish gills and lead to death, especially in the case of

large settled particles as they increase the nitrogenous compounds and stress the cultured fish

(Akinwole et al., 2016).

(b) Dissolved Wastes

Dissolved wastes are products of food metabolism in fish or decomposed, uneaten feed. In

dissolved wastes, the two major components of concern are nitrogen (N) and phosphorus (P)

products. These two elements constitute important components of protein, which is the main

component of fish feed. Fish, irrespective of species, require a high dietary crude protein

ranging from 25 to 50%. The high protein fish feeds contain high amounts of nitrogen and

phosphorus, yet less than 50% of these potential water pollutants (nitrogen and phosphorus)

are retained in the body of the fish (Piedrahita, 2013). Hence, a large percentage is transferred

into the culture water, where it becomes a nuisance, and, when finally released, have a lot of

environmental impacts. The nitrogen is mainly excreted in dissolved form as ammonia, while

phosphorus is excreted as particulate matter. Cultured fish have varying tolerances of

ammonia-nitrogen which depends on fish species, age, and physiological status. Warm water

fish are more tolerant than cold water fish, while adult fish are more tolerant than the

fingerlings and juveniles. Ammonia is generally recommended to be below 1mg/L in culture

tanks (Ajani et al., 2011).

(c) Microbial Wastes

The fish, their biotic and abiotic environments are inextricably linked, and changes in one

may reflect and affect the other (Wurt, 2000). Many microorganisms in pond water or water

used in intensive fish rearing could be pathogenic or opportunistic pathogens to fish, humans,

and planktons (Zmyslowska et al., 2003). These contaminating microorganisms have been

linked to poor water quality, which can be traced back to contaminated water sources and

high stocking densities (Sule et al., 2016). Water sources for earthen ponds are typically

untreated surface water runoffs from streams, rivers, lakes, and stored waters, whereas most

concrete ponds use an underground water source. Water is pumped from underground

sources, such as a well, and stored in a storage tank, which is used to refill the ponds when

the water needs to be changed every 3-5 days. The fish feed used in these ponds contains

organic materials and introduces a wide range of microorganisms.

According to Omojowo & Omojasola (2013) isolated six bacterial pathogens from cow dung

used as feed in the fish pond: E. coli, E. coli 0157:H7, Shigella dysenteriae, Staphylococcus

aureus, Salmonella typhi, and Aeromonas hydrophila. A cultivated fish's microbial flora

reflects its aqueous environment.

Freshwater fish in ponds commonly suffer from bacterial diseases such as various kinds of

skin ulcerations, albinoderma, erythroderma, furunculosis, and verticle-scale disease,

primarily caused by Aeromonas sp. and Pseudomonas sp. Some of these diseases were

reported to be most severe during the dry season, when declining water quality is a problem.

In Arizona, significant mortalities of cultured tilapia resulted from infection with a salt-
tolerant strain of the bacterium Aeromonas hydrophilia. Some prevalent pathogenic bacteria

of milkfish and tilapia isolated in Taiwan include species of the genera: Aeromonas,

Edwardsiella, Flavobacterium, Pseudomonas, and Streptococcus (Sule et al., 2016).

2.6 Impacts of wastewater effluents

The quality of wastewater effluents is responsible for the degradation of the receiving water

bodies, such as lakes, rivers, streams, etc. The potential deleterious effects of polluted

wastewater effluents on the quality of receiving water bodies are manifold and depend on

volume of the discharge, the chemical and microbiological concentration/ composition of the

effluents. It also depends on type of the discharge for example whether it is amount of

suspended solids or organic matter or hazardous pollutants like heavy metals and

organochlorines, and the characteristics of the receiving waters (Owili, 2003). Eutrophication

of water sources may also create environ- mental conditions that favour the growth of toxin-

producing cyanobacteria. Chronic exposure to such toxins produced by these organisms can

cause gastro- enteritis, liver damage, nervous system impairment, skin irritation and liver

cancer in animals (EPA, 2000; Eynard et al., 2000; WHO, 2006). In extension, recreational

water users and anyone else coming into contact with the infected water is at risk (Resource

Quality Services, 2004). The potential deleterious effects of pollutants from sewage effluents

on the receiving water quality of the coastal environment are manifold and depend on volume

of the discharge, the chemical composition and concentrations in the effluent (Owili, 2003).

2.6.1 Public health risk

Diseases caused by bacteria, viruses and protozoa are the most common health hazards

associated with untreated drinking and recreational waters. The main sources of these

microbial contaminants in wastewater are human and animal wastes (WHO, 2006). These
contain a wide variety of viruses, bacteria, and protozoa that may get washed into drinking

water supplies or receiving water bodies (Kris, 2007). Microbial pathogens are considered to

be critical factors contributing to numerous waterborne outbreaks. Many microbial pathogens

in wastewater can cause chronic diseases with costly long-term effects, such as degenerative

heart disease and stomach ulcer. The density and diversity of these pollutants can vary

depending on the intensity and prevalence of infection. The detection, isolation and

identification of the different types of microbial pollutants in wastewater are always difficult,

expensive and time consuming. To avoid this, indicator organisms are always used to

determine the relative risk of the possible presence of a particular pathogen in wastewater

(Paillard et al., 2005). Viruses are among the most important and potentially most hazardous

pollutants in wastewater. They are generally more resistant to treatment, more infectious,

more difficult to detect and require smaller doses to cause infections. Because of the

difficulty in detecting viruses, due to their low numbers, bacterial viruses (bacteriophages)

have been examined for use in faecal pollution and the effectiveness of treatment processes to

remove enteric viruses (Okoh, et al., 2007).

Bacteria are the most common microbial pollutants in wastewater. They cause a wide range

of infections, such as diarrhea, dysentery, skin and tissue infections, etc. Disease-causing

bacteria found in water include different types of bacteria, such as E. coli O157:H7; Listeria,

Salmonella, Leptosporosis, Vibrio, Campylobacter, etc (Absar, 2005). Wastewater consists of

vast quantities of bacteria, most of which are harmless to man. However, pathogenic forms

that cause diseases, such as typhoid, dysentery, and other intestinal disorders may be present

in wastewater. The tests for total coliform and faecal coliform nonpathogenic bacteria are

used to indicate the presence of pathogenic bacteria (EPA, 1996; APHA, 2001).

Wastewater effluents have been shown to contain a variety of anthropogenic compounds,

many of which have endocrine-disrupting properties. Reports have shown that exposure to
wastewater treatment effluents containing estrogenic chemicals can disrupt the endocrine

functioning of aquatic life, thus can cause permanent alterations in the structure and function

of the reproductive system (Liney et al, 2006). Evidence obtained from laboratory studies has

revealed the potential of several environmental chemicals to cause endocrine disruption at

environmentally realistic exposure levels. In aquatic environment, such effects have

reportedly been observed in mammals, birds, reptiles, fish, and mollusks from Europe, North

America, and other areas. The observed abnormalities in these groups of animals vary from

subtle changes to permanent alterations, including disturbed sex differentiation with

feminized or masculinized sex organs, changed sexual behavior, and altered immune function

(Vos et al., 2000). While multiple laboratory studies have shown the effects of such

compounds on an individual basis at elevated concentrations, little research has attempted to

characterize the effects of exposure to environmentally relevant mixtures of endocrine

disruptors (Vos et al., 2000; Sower, 2009).

2.6.2 Impact in Water Body

Water in catfish ponds usually has higher concentrations of nitrogen, phosphorus, organic

matter and biochemical oxygen demand than natural surface waters in the vicinity.

Concentrated aquatic animal production (CAAP) facilities such as hatchery and fish ponds to

mention few are major sources of wastewater effluent that contain high level of oxygen

demanding waste, producing objectionable odor in the receiving adjacent streams which most

fish farmer consider an ease waste disposal method. However, indiscriminate disposal of

untreated wastewater from fish pond on streams results in over- enrichment of water body

with nutrients causing eutrophication harmful to the aesthetic value of water body, preventing

sunlight penetration and decay of algae weeds which add odorous compound to the aquatic

system (Boyd, 2015).

2.7 Fish Pond Wastewater Treatment Methods

Wastewater treatment is a process to renovate wastewater before its reuse or discharge. The

goal is to reduce or remove organic matter, solids, nutrients, disease causing organisms and

other pollutants from wastewater. A number of physical, chemical, and biological methods

used in wastewater treatment have been applied in aquaculture systems (Cao and Wang,

2010).

(a) Mechanical Filtration

Mechanical filtration can remove suspended solids as well. There are several types of

filtration, including drum filtration, screen filtration, and sand filtration. These filters have

mesh sizes as small as 40 um, but due to the large amount of wash water required, filters with

mesh sizes of 70 um or larger are usually preferred. Small suspended solids tend to

accumulate in recirculating systems despite these highly improved filtration methods (Cao

and Wang, 2010). Small suspended solids can be removed using either chemical or biological

oxidation. Foam fractionation is also categorized as a type of mechanical filtration. Through

air-stripping, it can remove and separate soluble organic substances and suspended solids

inside the rising air bubbles. Foam fractionation can prevent accumulation of the toxic

substances in the aquaculture wastewater.

(b) Aeration

Aeration is widely used in most rural areas of China to provide oxygen to the effluent being

treated as well as to remove odorous gases from bottom sediment. Some methods for

bringing water into contact with air include allowing it to fall down a set of steps, splashing

and breaking up into films and drops; spraying it into the air; and blowing or drawing air

bubbles through it. Pumping by air lift has a minor aerating effect. After exchanging the
oxygen-rich surface water with the pond's bottom water, some organic-rich sediment could be

effectively decomposed.

(c) Chemical Process

Chemical removal is a wastewater treatment method that involves the addition of chemicals

to form particles that settle and remove contaminants. Chemical treatment is still an important

part of many wastewater treatment plans. The general goals of chemical treatment are as

follows: removal of suspended solids (turbidity) from water; pH adjustment; removal of

dissolved material in water; and improvement of water quality. Coagulation/flocculation,

chlorination, chloramination, ozonation, and ultraviolet light (UV) are the most commonly

used chemical treatment methods (Gray, 2015). Flocculation reduces pathogen levels while

also removing particles that could protect pathogens from chemical or thermal destruction, as

well as organic matter that could wind up chlorine added for purification (Akpor, 2011).

Adding coagulation chemicals such as alum will then increase the rate at which the

suspended particles settle out by combining many smaller particles into larger floc which will

settle out faster. In bulk water treatment, the alum dose is varied until the required dose is

found. Chlorination is the most prevalent practice of disinfection. Although it requires a

relatively long contact time, because of its high oxidation potential, it is still a disinfectant of

choice. However, chlorine does not only disinfect, but also rapidly reacts with contaminants

such as NH4+, NO2-, H2S, Fe2+ and other organic compounds, thus leading to the formation

of compound called trihalomethanes, which are considered health hazards Chloramination

(use of chloramines as disinfectant) has the benefit of reduction of formation of

trihomethanes but because of their relatively longtime after discharge to receiving water

environment, always lead to toxicity problems. It is mostly used in treatment of wastewater

with high organic compounds (Tchobanoglous and Kreith, 2016). Ozonation is primarily

employed in secondary wastewater treatment. It has good bactericidal and virucidal

properties, is colorless, and does not produce toxic byproducts. Its disadvantages include its

high cost and lack of maintenance. Because there is always the possibility of microbial re-

growth in the water after treatment. UV and chlorine have been shown to be effective in

wastewater disinfection. Ultraviolet light does not produce toxic byproducts, and its faecal

indicators are extremely sensitive. Its drawbacks are that it is costly, increases the volume of

sludge produced, and usually results in sludge with poor dewatering and settling

characteristics. The main advantages of chemical treatment over biological processes are:

mineralization of non-biodegradable compounds and smaller reactor volume (Tchobanoglous

and Kreith, 2016).

2.8 Empirical Review

Adedayo and Anthony, (2014), carried out a study on Bacteriological study of pond water for

aquaculture purposes, two objectives guided the study. Bacterial isolated were purified into

pure culture and identified base on their morphological, cultural and biochemical test using

standard microbiology procedure. This study shows diversified forms of bacterial species

populating the pond water sources. This includes Staphylococcus spp., Streptococcus spp.,

Bacillus spp., Pseudomonas spp., E. coli, Enterobacter spp., Proteus spp., Citrobacter spp.

and others.

Njoku, Agwa, and Ibiene, (2015), carried out an investigation on the microbiological and

physicochemical profile of some fish pond water within the Niger Delta region of Nigeria.

The mean total heterotrophic bacteria count was higher in concrete ponds (6.5x10 5 to 7.4x105

cfu/ml) than (6.3x105 to 6.5x105 cfu/ml) in earthen pond and the total coliform was higher in

earthen pond (7.3x107 to 2.3x107 cfu/100ml) than concrete pond (5.9x10 7 to 2.1x107

cfu/100ml). The study revealed that the ponds were grossly contaminated with pathogenic

microorganisms which poses a risk to human health, thus of significant public health concern.
Ogeneogaga, and Solomon, (2017), carried out study on Physico-Chemical and

bacteriological Investigation of Selected Fish Pond in Kuje Area Council, Nigeria, to evaluate

the water quality of the ponds. The frequencies of occurrence of isolated bacterial species

were as follows: E. coli 25%, Flavobacterium spp. 16.7%, Staphylococcus spp. 16.7%.

Bacillus spp. 16.7%, Psuedomonas spp. 8%, Samonella spp. 8%, and Bacillus cereus 8%.

The presence of this organism show a lack of tentative pond management services which

could be harmful to fishes and humans. There is therefore a need to monitor water quality and

detect the actual source of contamination and subject the water through a form of treatment to

prevent an epidemic outbreak.

 CHAPTER THREE

MATERIALS AND METHODS

3.1 Study Area

The study was carried out in Calabar Metropolis to determine the bacteriological quality of

fish pond effluent from selected fish ponds

3.2 Collection of Samples

The fish pond wastewater was collected at 13 different locations in Calabar metropolis; all

sample were collected with their control. The fish pond wastewater samples were collected at

15cm deep into a sterile container and labelled correctly. The samples were covered and

immediately transported to Microbiology laboratory of University of Cross River State for

bacteriological analysis.

3.3 Preparation of Media

All media were aseptically prepared according to the manufacturer instructions. The media

used were Nutrient Agar, Macconkey Agar, Simmons Citrate Agar, and Peptone water.

Preparation of Nutrient Agar: 5.6g of Nutrient agar powder was weighed and mixed into

280ml of distilled water in a 500ml conical flask. The content of the flask was rotated and

heated for proper dilution of the media. The flask was covered with cotton wool, wrapped

firmly with aluminum foil and autoclaved at 121 0C for 15minutes. It was allowed to cool at

450C before it was dispensed aseptically in 20ml volume of petri dishes. The medium was

allowed to gel on petri dishes.

Preparation of Macconkey Agar: 25.4g of Macconkey agar powder was weighed and

mixed into 1274ml of distilled water in a 1000ml and 300ml conical flask. The content of the

flask was rotated and heated for proper dilution of the media. The flask was covered with

cotton wool, wrapped firmly with aluminum foil and autoclaved at 121 0C for 15minutes. It
was allowed to cool at 45-500C. It was mixed well before pouring into sterile petri dish of

20ml volume and the medium was allowed to gel on the petri dishes.

Preparation of Citrate Agar: 3.1g of Citrate Agar powder was weighed and mixed into

629ml of distilled water in a 1000ml conical flask. The content of the flask was rotated and

heated for proper dilution of the media. 5ml of the dissolved media was dispense into

different test tubes under aseptic conditions; plugged firmly with cotton wool and wrapped

with foil paper, it was then autoclave at 121 0C for 15minutes. It was then removed, slanted

and allowed to cool and solidify before being used.

3.4 Preparation of Samples

1ml of each sample was added to first 10ml of sterile distilled water in test tube and mixed

gently. 1ml of the dilution was serially diluted to obtain 10 -1 dilution factor. 0.5ml of 10-5 was

used for isolation of bacteria using the spread plate method. The plates were then incubated at

370C for 24 hours. After incubation bacteria counts were determined using colony count

machine and expressed in CFU/g.

3.5 Purification of Bacteria Isolate

Pure cultures were obtained by sub-culturing distinct colonies onto sterile nutrient agar plates

severally. The pure cultures obtained were then transferred onto agar slants in EDTA bottles

and incubated at 270C. The bottles are then stored in the refrigerator.

3.6 Characterization and identification of isolates

The bacterial species isolate was identified and also characterized after investigating their

Gram reaction, their physiological characteristics and cell micro-morphology. Morphological

characterization was based on the colony morphology which includes, colour elevation,

consistency and moisture contents (wet or dry colony).

Gram Staining: A drop of distilled water was placed on a grease free slide and a fresh

culture of 24hrs was smeared on the slide with the aid of a sterile inoculating wire loop. The
smear was covered with crystal violent (primary stain) for 60 seconds. It was rapidly washed

off under running water for 5 seconds. The smear was covered with Lugol’s iodine (which

serves as mordant) after tipping off all the water. The Lugol’s was allowed to stand for 60

seconds, rinsed again in slowly running water. Alcohol which serves as decolorizer was drop

for 5 seconds and immediately rinsed off. The smear was covered with safranin reagent

(secondary stain) for 30 seconds. The slide was washed off slowly under running tap. Air

dried and then viewed/examined under the microscope using 100x objective oil immersion

lens. Gram Negative bacteria appeared purple or violet while Gram Negative Bacteria

appeared red or pink.

Catalase Test: A 24 hour old culture was used to carry out the test. The sterile wire loop was

used to make a homogenous suspension on the slide. A drop of hydrogen peroxide (H 2O2)

was added to the suspension and the occurrence of effervescence indicate a Positive reaction

while its absence indicates a Negative reaction.

Oxidase Test: This test was carried out using freshly prepared oxidase reagent. After placing

filter paper in a clean petri dish, 2 to 3 drops of oxidase reagent (1% of aqueous solution of

tetra methyl phenylene diamine dihydrochloride) was placed on filter paper. With the aid of a

sterile wooden rod, a colony of each of the isolates was picked and smeared on the filter

paper in the petri dish. The development of blue-black colour within few seconds on filter

paper indicate positive results.

Citrate Utilization Test: This test showed the ability of some bacteria to utilize citrate as

their sole carbon source. Simmon’s citrate medium formulation includes sodium citrate as

sole source of carbon, ammonium phosphate as the sole source of nitrogen and the pH

indicator bromothymol blue. Slants of Simmon’s citrate were prepared in the test tubes
according to the manufacturer’s instruction. Using a sterile straight wire loop, the slants were

inoculated lightly and were incubated at 370C for 72 hours. Colour change (green to blue)

indicate utilization of Citrate by the organism.

Indole Test: This test determines the ability of an organism to produce indole from the

degradation of amino acid tryptophan. Tryptophan is hydrolyzed by tryptophanase to produce

three possible end products – one of which is indole, the others are pyruvate and ammonium

ion. Place several drops of indole spot reagent on a piece of filter paper, with an inoculating

loop or wooden applicator stick, pick a portion of a 24 hour isolated colony from a non-

selective media and rub it onto the reagent saturated area of the filter paper and examine

immediately. Pink or red colour indicate Positive result while no colour change indicate

negative result.

Motility Test: This test is to use determine if an organism is motile or non-motile. Pick a
1/3
pure colony of 24hrs and stab once to a depth of only to ½ inch in the middle of the tube.

Be sure to keep the needle in the same line it entered as it is removed from the medium.

Incubate at 35o – 37oC and examine daily for up to 7 days. Observe for a diffuse zone of

growth flaring out from the line of inoculation. Diffuse, hazy growths that spread throughout

the medium rendering it slightly opaque indicates Positive result while growth that is

confined to the stab-line, with sharply defined margins and leaving the surrounding medium

clearly transparent indicates negative result.

Coagulase Test: Coagulase test is used to differentiate staphylococcus aureus which convert

fibrinogen to fibrin (positive) from S. epidermis and S. saprophyticus (negative). A slide test

was carried out to determine bound coagulase. Place a drop of physiological saline on each

end of a slide, or on two separate slides, with the help of wire loop or wooden stick, emulsify

the isolated colony in each drops to make two thick suspensions. Add a drop of human
plasma to one of the suspensions, and mix gently. Look for clumping of the organisms within

10 seconds. No plasma is added to the second suspension to differentiate any granular

appearance of the organism from true coagulase clumping.

Triple sugar iron agar (TSI)

This test is generally used for the identification of enteric bacteria. It is also used to

distinguish the Enterobacteriacieae, from other gram negative intestinal bacilli by ability to

catabolize, glucose, lactose or sucrose. About 65g of Triple sugar iron agar was weighted and

mixed into 100ml of distilled water in a conical flask and autoclaved at 121 0C for 1 hour. The

agar was poured into test tubes and slant them to solidify. With the aid of sterile wire loop a

loopfull of the organisms was picked from the stock cultures and inoculated by streaking on

the slant surface and then stabbed into the butt.

The tubes were sealed with foil paper and incubated for 18 – 24 hours at 37 0C. After 24 hours

the tubes were examined for colour and gas production.

 CHAPTER FOUR

RESULT

4.1 Total heterotrophic bacterial count and total coliform count

Table 4.1 shows the results of the bacteriological analysis of 13 samples of fish farm effluent

at different location in the study area shows. The total heterotrophic bacterial count ranges

from 1.5x104 cfu/100 ml to 7.0x104 cfu/100ml, and total coliform from 1.6x104 cfu/100ml to

5.2x104 cfu/100ml. UNA sample had the highest value of the bacterial count of 7.0 x10 4 and

TFAD have the lowest coliform count of 1.6 x104.

4.2 Morphological and biochemical characterization of bacterial isolated from fish farm

effluent

Based on the morphological and biochemical characteristics of bacteria isolated from fish

farm effluent as presented in table 4.2, the following bacteria were isolated; Staphylococcus

aureus; Enterococcus spp; Klebsiella spp; Salmonella spp; Pseudomonas aeruginosa;

Proteus spp; Staphylococcus epidemidis. The isolates were differentiated on the basis of the

cultural and morphological studies as well as the biochemical test.

4.3 Percentage occurrence of bacterial isolates from fish farm effluents

Table 4.3 shows the percentage of occurrence of bacterial isolate of 13 samples from fish

farm effluent in Calabar Metropolis. The organism with the lowest occurrence in the studied

samples was Enterococcus spp 7.6%, Klebsiella spp 11.5%, Pseudomonas aeruginosa 11.5%,

Staphylococcus epidermidis 11.5%, Staphylococcus aureus 15.3%, Proteus spp 19.5%, and

Salmonella spp 23.0%, have the highest occurrence.

 Table 4.1.

Total heterotrophic bacterial count and total coliform count (cfu/ml) x103

Sample Total Heterotrophic Total coliform

Code Bacterial count count
NMSMA 1.5 x104 2.3 x104

TFAD 2.0 x104 1.6 x104

EBIA TNTC 2.1 x104

EISOM 3.4 x104 2.0 x104

UOPC 4.2 x104 2.5 x104

ABAC 5.0 x104 3.7 x104

OECM 6.3 x104 5.2 x104

AAA 4.7 x104 2.9 x104

UNA 7.0 x104 4.4 x104

UWU 3.9 x104 3.6 x104

PAS 2.2x104 2.0x104

ENI 4.3X104 3.3X104

AFJ 1.8X104 1.7X104

Note: NMSMA -Nelson Mandela Street, TFAD - Atakon Drive, EBIA- Eburutu Barrack
EISOM - Eteta Ita Street, UOPC- Umoa Okon, ABAC- Access by Atimbo, OIRM-98 Old
Ikang Road, OECM-8 Offiong Ekpo Close, AAA- Anatigha, UNA – Umo Orok, PAS- Palm
Street, ENI- Ene-Ita, AFJ- Afokang Jebs
 Coagula
Oxidase

Possible
Motility

Catalas

organis
Citrate
Sample Gram

Indole
Shape Surface Colour Elevation
S/N stain

m
se
e
NMSMA Circular Smooth Creamy Raised +ve strep. - + - - - - Enterococcus spp.
TFAD Irregular Glistering Creamy Raised -ve rods - + - + - - Klebsiella spp.
EBIA Circular Smooth Yellowish Flat -ve rods + + - + + - Salmonella spp.
EISOM Circular Smooth Yellowish Raised -ve rods - - - + - + Staphylococcus aureus
UOPC Circular Smooth White Convex -ve rods + + + + - - Pseudomonas auruginosa
ABAC Contoured Smooth Creamy Flat -ve rods + - - + + - Proteus spp.
OECM Circular Smooth White Flat +ve rods - - + + - - Staphylococcus epidemidis
AAA Circular Smooth White Flat +ve rods - - + + - - Staphylococcus epidemidis
UNA Contoured Smooth Creamy Flat -ve rods + - - + + - Proteus spp.
UWU Circular Smooth White Convex -ve rods + + + + - - Pseudomonas auruginosa
PAS Circular Smooth Yellowish Raised -ve rods - - - + - + Staphylococcus aureus
ENI Circular Smooth Yellowish Flat -ve rods + + - + + - Salmonella spp.
AFJ Irregular Glistering Creamy Raised -ve rods - + - + - - Klebsiella spp.
NMSMA Circular Smooth Creamy Raised +ve strep. - + - - - - Enterococcus spp.
TFAD Circular Smooth Yellowish Flat -ve rods + + - + + - Salmonella spp.
EBIA Circular Smooth Yellowish Raised -ve rods - - - + - + Staphylococcus aureus
EISOM Circular Smooth White Convex -ve rods + + + + - - Pseudomonas auruginosa
UOPC Contoured Smooth Creamy Flat -ve rods + - - + + - Proteus spp.
ABAC Circular Smooth White Flat +ve rods - - + + - - Staphylococcus epidemidis
OECM Circular Smooth Yellowish Flat -ve rods + + - + + - Salmonella spp.
AAA Contoured Smooth Creamy Flat -ve rods + - - + + - Proteus spp.
UNA Circular Smooth Yellowish Raised -ve rods - - - + - + Staphylococcus aureus
UWU Circular Smooth Yellowish Flat -ve rods + + - + + - Salmonella spp.
PAS Circular Smooth Yellowish Flat -ve rods + + - + + - Salmonella spp.
ENI Contoured Smooth Creamy Flat -ve rods + - - + + - Proteus spp.
AFJ Irregular Glistering Creamy Raised -ve rods - + - + - - Klebsiella spp.
Table 4.2. Morphological and Biochemical characterization of Bacteria Isolated from Fish Pond Effluent
 Table 4.3

Percentage occurrence of bacterial isolates from fish farm effluents

Bacterial isolates Frequency (F) Percentage (%)

Staphylococcus aureus 4 15.3

Enterococcus spp 2 7.6

Klebsiella spp 3 11.5

Salmonella spp 6 23.0

Pseudomonas aeruginosa 3 11.5

Proteus spp 5 19.2

Staphylococcus epidemidis 3 11.5

Total 26 100
 CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion

The bacteriological examination of fish pond effluent is important as this creates tentative

pond management services which could be harmful to fishes and humans. The results of the

bacteriological analysis of 13 samples of fish farm effluent at different location in the study

area shows the total heterotrophic bacterial count result ranges from 1.5x10 4 cfu/100 ml to

7.0x104 cfu/100ml, and total coliform from 1.6x104 cfu/100ml to 5.2x104 cfu/100ml. UNA

had the highest value of the bacterial count of 7.0 x10 4 and TFAD have the lowest coliform

count of 1.6 x104 which is the lowest. This result is in line with the study of Njoku, Agwa,

and Ibiene, (2015), and Adedayo and Anthony, (2014).

Based on the morphological and biochemical characteristics of bacteria isolated from fish

farm effluent, the following bacteria were isolated; Staphylococcus aureus; Enterococcus

spp; Klebsiella spp; Salmonella spp; Pseudomonas aeruginosa; Proteus spp; Staphylococcus

epidemidis. This findings is slightly similar to Ogeneogaga, and Solomon, (2017), who

reported the presence of E. coli, Flavobacterium spp., Staphylococcus spp., Bacillus spp.,

Psuedomonas spp., Salmonella spp., and Bacillus cereus in a similar study. Based on the

gram reaction of bacterial isolates, Gram negative bacteria dominated with the total of 5

Gram negative bacterial and two Gram positive bacteria.

Considering the prevalence of occurrence, Salmonella species had the highest prevalence of

occurrence while Enterococcus spp. had the least. The presence of the pathogenic organisms

such as Salmonella typhi, E. coli, Enterococcus spp. is an indication that the fish ponds were

grossly contaminated and called for public concern.

5.2 Conclusion

This study suggests that there is need to monitor the quality of wastewater that is being

discharged from the fish ponds into the environment. Hence, its release and use should be

with caution and subjected to closer regulation by environmental, agricultural and health

protection agencies. It is therefore recommended that microbiological examination and

physicochemical analysis of the discharged wastewaters from fish ponds be carried out

regularly for proper monitoring so as to prevent avoidable epidemics in the country. Good

quality waters such as well or borehole should be used in the fish pond rather than water from

questionable sources such as river, stream, and surface runoff. The installation of waste water

management techniques should be approved and enforced by the government on all fish farm

owners. Moreover, public awareness on the effects caused by the indiscriminate release of

fish ponds’ effluents into the surroundings should be organized for farmers especially those

in the rural areas. And Fish feeds should be obtained from reliable manufacturers to reduce

the level of contamination associated with feeds.

5.3 Recommendation

Based on the outcome of this study, the following recommendations were made:

1. The result showed that there was no fish pond water sample that was free from

bacteria and fungi, an indication that the entire fish pond water samples were

contaminated by microorganisms.

2. The contamination could have arisen from different sources which include air, source

of water and fish feeds could have been responsible for the introduction of these

organisms into the fish pond. Also, it was shown in this study that the gamma

distribution provided best fit for the microbial count.

3. Sanitary conditions under which fish are reared in ponds should be improved, by

following standard or good practices; such as use of good quality water, use of feeds
 with high microbial quality, regular draining of pond water after specific period of

time, closure of ponds to the public among other things.

4. Good quality water such as borehole should be used in the fish pond rather than water

from questionable sources such as river, stream, and surface-runoff.

5. Waste water should be treated either by physical methods which involve filtration

through slow sand filters, rapid sand filters, sand-beds or chemical methods such as

addition of disinfectants e.g. chlorine before final disposal into surrounding drains.