2. diarrheal diseases; 90% of these deaths are among
children, mostly in developing countries. Bloomfield
et al. (2009) concur that diseases resulting from
contaminated water comprise 80% of the total
disease burden in some poor countries. The WHO
(Anonymous 2004a) estimates that one-third of
deaths in developing countries are caused by
consumption of contaminated water, and on
average as much as one-tenth of each person’s
productive time is sacrificed to waterborne diseases.
Likewise, the WHO (Anonymous 2008b) estimates
that up to half of all hospital beds in the world are
occupied by victims of water contamination.
Although clean water is a valuable natural
resource essential for human health and ecological
integrity, most fresh water bodies are increasingly
being degraded by anthropogenic activities, thus,
exposing the water users to a higher risk of
waterborne diseases (Bloomfield et al. 2009).
Unfortunately, important water quality concerns
persist in most developing countries in Africa as a
result of increased livestock and anthropogenic
activities. Access by livestock and people to river
banks is very common in most developing countries,
particularly among the poor rural communities or
those living in informal urban settlements where
most households do not have access to tap water.
Visits to rivers are often periodic but frequent
especially during dry periods when alternative
sources of water such as groundwater and dams are
irregular, lacking or inaccessible. During such visits,
several activities are performed, among them
livestock watering, bathing, swimming, waste
disposal, washing of clothes and vehicles among
others, which often contribute to water quality
degradation (Yillia et al. 2009). The in-stream
anthropogenic activities usually constitute a major
source of diffuse pollution that can influence
nutrient levels and water quality (Zamxaka et al.
2004). Insufficient communal sanitation facilities
such as toilets especially in informal settlements of
developing countries in Africa often give way to
open defecation along the river banks, in drains and
open spaces. This results in fecal matter being
intermixed with household refuse that finally ends
up in aquatic ecosystems through surface runoffs.
Many urban centers in Africa either lack sewerage
systems or operate inefficient systems that serve
only a small proportion of the urban population
(Anonymous 1996). Even where sewers exist they
are often blocked leading to overflow of raw sewage
into streets and open spaces, providing suitable
grounds for disease-causing pathogens. Such
discharges can significantly compromise water
quality in aquatic systems, which may pose serious
health risks, more so when the water is meant for
domestic use (Anonymous 2004b).
Rapid population growth and urbanization amid
economic stagnation in Kenya have resulted in an
increased proportion of people living in absolute
poverty in urban and peri-urban areas (Anonymous
2003b). A study on sanitation and hygiene among
women living in informal urban settlements by the
Central Bureau of Statistics (Anonymous 2003b)
showed that only 42% of the women disposed of
children fecal matter hygienically into a toilet or
latrine. The rest either rinsed it away in rivers or
threw it outside their dwelling place intermixed with
other household waste. Poor human waste disposal
methods and fecal droppings from livestock are
routes through which fecal matter can gain entry
into aquatic systems, thus introducing pathogens,
nutrients and organic matter (Vikaskumar et al.
2007).
Studies by WHO/UNICEF (Anonymous 2004b)
showed that sewage is the largest contaminant of
water masses around the world causing increased
microbial contamination, nutrient enrichment and
subsequent eutrophication of surface waters as was
also observed by Kanu and Achi (2011). Byamukama
et al. (2005) acknowledged that fecal matter may
present a significant health risk to the public. Scott
et al. (2002) on the other hand reported that the
level of risk depends on the origin and level of
contamination. Human excreta are regarded as a
major risk to public health as they often contain
human-specific enteric pathogens. Microbial
contamination of surface waters can also trigger the
spread of many other infectious diseases such as
cholera, dysentery, typhoid, hepatitis,
cryptosporidiosis, ascariasis, and even
schistosomiasis among others (Anonymous 2000).
Anyona et al.
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EurAsian Journal of BioSciences 8: 1-11 (2014)
3. Mulot and Bomet urban centers, like many other
centers in Kenya, are experiencing rapid population
growth characterized by poor urban planning,
informal settlements, limited amenities, poor waste
handling capacity, poor sanitation and lack of
sewage treatment facilities (Anonymous 2007a). The
two towns also differ in size and population. This
implies that the Mara River tributaries serve not only
as critical sources of water for domestic use for the
locals, but also as conduits for waste and raw
sewerage that often contaminate water resources
(Karani and Mutunga 2004). However, the extent to
which the waters are polluted and the potential risk
they pose to human health cannot be ascertained
without focused analysis to determine pollutant
levels. This study sought to assess pollutant levels
by use of fecal indicators of water quality including
total coliform and Escherichia coli as well as nutrient
levels (total nitrogen and total phosphorus). Since
half of the world’s population (47%) lives in urban
areas (Anonymous 2002a), many rivers that flow
through such areas are often highly polluted, more
so those flowing through informal settlements. This
study will therefore be of general importance and
can be widely applied to other river systems faced
with similar challenges to those of the Mara River.
Study area
The Mara River Basin lies between longitudes 33°
88' E and 35° 90' E and latitudes 0° 28' S and 1° 97' S,
at altitudes of between 2,932 m at the source in the
Mau Forest Escarpment to 1,134 m at the discharge
point into Lake Victoria (Fig. 1). It covers a surface
area of about 13,504 km2
(Mati et al. 2008). The two
perennial tributaries, Amala and Nyangores,
originate from the western Mau escarpment in the
Rift valley in Kenya and flow through the towns of
Mulot and Bomet, respectively, before forming a
confluence and flowing further down as the Mara
River. This eventually discharges its waters into Lake
Victoria at Mosirori swamp in Musoma in Tanzania
(Mati et al. 2008).
Research design and sampling sites
This descriptive cross-sectional study was carried
out between the 4th
and 8th
of July in 2011. The
sampling sites were selected based on their
characteristics and location along the two perennial
Mara River tributaries of the Amala and Nyangores.
In total, eight sampling sites were chosen; four along
each tributary. The sampling sites on each tributary
were distributed as follows; three within the towns
(Bomet town - along the Nyangores tributary and
Mulot town - along the Amala tributary) to capture
the influence of anthropogenic activities within
urban centers and a fourth site located at the upper
catchment spring, that discharges its water into each
tributary to act as a control. Water samples were
collected for nutrients and coliform bacterial
analysis in replicates of three and five, respectively,
from each sampling site.
Determination of total phosphorus and total
nitrogen
Total Nitrogen (TN) and Total Phosphorus (TP)
were determined on unfiltered water samples
following the methods outlined in APHA-AWWA
(Anonymous 2005). Total nitrogen was determined
using the persulfate digestion method, while total
phosphorus was determined by the ammonium
molybdate method.
Determination of total coliform and
Escherichia coli
Five replicate water samples for coliform
bacterial determination were collected from below
the water surface at intervals of 10 m along each
tributary using sterile 250 mL glass bottles. The
bottles were inverted downwards against the water
current, with the hand kept downstream from the
neck to avoid contamination. The samples were
stored in an ice-packed box and delivered within the
six-hour holding time to the Longisa Sub-District
Hospital Microbiology laboratory for microbial
analyses. Coliform abundance in the water samples
was estimated using the most probable number
(MPN) procedure by the multiple tube fermentation
technique, which involved three successive steps,
namely presumptive, confirmed and completed
tests (Feng et al. 2002).
A summary of the resulting characteristics of the
data were given by descriptive statistics presented
as means and standard deviations, while variation in
3
Anyona et al.EurAsian Journal of BioSciences 8: 1-11 (2014)
MATERIALS AND METHODS
4. nutrient and coliform bacterial levels between sites
along the Amala and Nyangores tributaries were
determined using One-way analysis of variance
(ANOVA), followed by post hoc separation of means
by use of the Duncan Multiple Range Test (DMRT), to
establish the significant differences between sites.
Regression analysis was used to describe
relationships between nutrients and coliform
bacteria, while the Student’s t-test was used to
deduce the possible differences in nutrients and
coliform levels between the Amala and Nyangores
tributaries. Statistical analyses were performed
using SAS V9.0 software. A P<0.05 was chosen as the
significance level.
Nutrient (phosphorus and nitrogen)
concentration
The mean total phosphorus was significantly
higher along the Amala tributary compared to the
Nyangores tributary (Student’s t-test, P= 0.02).
However, total nitrogen levels did not show any
significant differences between the two tributaries.
Considering each tributary separately, the mean TN
levels varied significantly between sites along the
Nyangores tributary (F (3,7)= 530.71, P<0.0001).
Further, DMRT confirmed that TN was significantly
higher (1967±6 μg/L) at the exit point from the town
of Bomet along the Nyangores tributary, but it was
lowest (1230±19 μg/L) at the upper catchment
spring (Fig. 2). Likewise, mean TP levels varied
significantly between sites along the Nyangores
tributary (F (3,7)= 77.47, P= <0.001). Significantly
higher TP levels (685±6 μg/L) were recorded at the
exit point from Bomet along the Nyangores
tributary, while the lowest (404±16 μg/L) was
recorded at the upper catchment spring, draining
into the same tributary (Fig. 3).
At the Amala tributary, the mean total
phosphorus levels varied significantly between sites
(F(3,7)= 28.83, P= 0.0036). The Duncan Multiple
Range Test further showed that mean TP levels were
significantly lower at the upper catchment spring
compared to other sites located within the
urbanized area (Mulot town) along the Amala
tributary (Table 1, Fig. 4). However, no significant
differences were observed in TN levels between
sites along the Amala tributary (P= 0.245). Even
though total nitrogen level was relatively higher
Anyona et al.
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EurAsian Journal of BioSciences 8: 1-11 (2014)
RESULTS
Fig. 1. Map of study area showing the location of the
sampling points along the Amala and Nyangores tributaries
of Mara River.
Fig. 2. Mean total nitrogen levels in water samples from
different sites along the Nyangores tributary, Kenya (n= 8).
Means with different superscripts are significantly different at
P<0.05.
Fig. 3. Mean total phosphorus levels in water samples from
different sites along the Nyangores tributary, Kenya (n= 8).
Means with different superscripts are significantly different at
P<0.05.
5. along the Amala than Nyangores tributary, the
difference was not significant (Student’s t-test, P=
0.06). Total nitrogen levels showed a steady increase
downstream along both the Amala and Nyangores
tributaries (Table 1, Fig. 5).
Total coliform bacteria (MPN/100 mL) along
the Nyangores and Amala tributaries
The mean total coliform level was 531 MPN/100
mL along the Amala tributary and 272 MPN/100 mL
along the Nyangores tributary (Fig. 6). One-way
ANOVA indicated significant variations in total
coliform bacteria between sites along the Nyango-
res tributary (F(3,19)= 6.91, P= 0.003). The Duncan
Multiple Range Test further showed site-specific
significant differences between the upper catch-
ment spring and those sites located within the
urbanized area (Bomet) along which the river flows.
As expected, total coliform bacterial levels increa-
sed downstream along the Nyangores tributary
(Table 2).
Along the Amala tributary, the highest mean total
coliform level (1100±0 MPN/100 mL) was recorded
at the middle point of Mulot, while the lowest
(225±220 MPN/100 mL) was at the upper catchment
spring that drains into the same tributary (Fig. 7).
There was a significant difference in total coliform
between sites along the Amala tributary (F(3,19)=
5.09, P= 0.012), with DMRT further indicating that
total coliform levels at the middle point of Mulot
was significantly higher than at all other sites (Table
2). The mean coliform levels between the two
tributaries were, however, not significantly different
(P= 0.103).
Escherichia coli levels along the Amala and
Nyangores tributaries
Escherichia coli levels varied significantly
between sites along the Nyangores tributary
(F(3,19)= 31.82, P<0.0001), with DMRT further
showing that E. coli levels at the upper catchment
spring were significantly lower compared to all other
sites located within Bomet along the Nyangores
tributary (Fig. 8). Generally, sections of the Nyango-
res tributary flowing through Bomet recorded
relatively high E. coli levels compared to the upper
catchment spring that discharges into the same
tributary. Along the Amala tributary, however,
higher E. coli levels (27.5%) were recorded at the
upper catchment spring compared to those
recorded at the lower sections of the tributary. E.
coli proportions showed a close range of between
23.5% and 27.5% across all the sites, and no
significant differences were observed between sites
along the tributary (P>0.05). Overall, regression
analysis on nutrients and E. coli showed that total
nitrogen (R2
= 0.6886, n= 8, P= 0.01) was predictive of
E. coli abundance along both tributaries combined,
but was not predictive of total coliform along the
Mara River tributaries.
5
Anyona et al.EurAsian Journal of BioSciences 8: 1-11 (2014)
Table 1. Total nitrogen and phosphorus levels along the
Amala and Nyangores tributaries.
*Means with different superscripts in the same column are
significantly different at P<0.05. (Data analyzed by Duncan’s
Multiple Range Test).
Table 2. Mean total coliforms (MPN/100 mL) along the
Amala and Nyangores tributaries.
*Means with different superscripts in the same column are
significantly different at P<0.05. (Data analyzed by Duncan’s
Multiple Range Test).
Fig. 4. Mean total phosphorus levels in water samples from
different sites along the Amala tributary, Kenya (n= 8).
Means with different superscripts are significantly different at
P<0.05.
6. This study was novel in evaluating the role of
anthropogenic activities on water pollution along
the Mara River. It is indicative that most polluted
sites were those frequented by livestock and people
for various purposes. Many factors and sources were
identified as contributors to the high bacterial levels
along the Mara River tributaries. Overdependence
on the Mara River waters by the communities living
along the riparian community exposes humans to
increased risk of contracting waterborne diseases.
Given the expected scale of urban population
growth in the coming decades, continued growth in
the number of urban poor will pose a fundamental
challenge to urban-flowing rivers around the world.
This will most likely expose more people, especially
those in developing countries, to increased risks of
waterborne diseases. The findings of this study are
therefore not only of local or regional interest but
can be generalized to other lotic systems in both
developing and developed countries, the majority of
which are now faced with more or less similar
challenges as those facing the Mara River Basin.
Nutrient (phosphorus and nitrogen) and
coliform bacterial levels
The Mara River tributaries, like many other rivers
worldwide, serve as valuable sources of water for
various uses, key among them being domestic use.
However, increased anthropogenic activities
continue to negatively impact on water quality
making it unsafe for human consumption. More
disturbed sites, especially those located within the
urbanized areas (Mulot and Bomet) along the Amala
and Nyangores tributaries, respectively, had higher
nutrients (total nitrogen and phosphorus) and also
total coliform levels compared to those located at
the upper catchment springs. This was a clear
indication of the potential contribution of
anthropogenic activities to nutrient enrichment and
Anyona et al.
6
EurAsian Journal of BioSciences 8: 1-11 (2014)
DISCUSSION
Fig. 5. Mean total nitrogen levels in water samples from
different sites along the Amala tributary, Kenya.
Fig. 6. Mean total coliform levels in water samples from
different sites along the Nyangores tributary, Kenya.
Means with different superscripts are significantly different at
P<0.05.
Fig. 7. Mean total coliform levels in water samples from
different sites along the Amala tributary, Kenya.
Means with different superscripts are significantly different at
P<0.05.
Fig. 8. Proportion of Escherichia coli levels along the Amala
and Nyangores tributaries, Kenya.
7. microbial pollution of the Mara River waters. In the
current study, the nutrient levels recorded ranged
between 430 μg/L and 832 μg/L for TP and 1230
μg/L and 2201 μg/L for TN across both tributaries.
These were almost similar to those recorded at the
Yarra River located in Victoria bay, Australia in a
longitudinal study (1994-2009). Precisely, the TN
levels ranged between 190 and 5150 μg/L, while TP
values between 100 and 870 μg/L in four of the five
stations sampled along the river. This was also the
case for the Mara River Basin where an increase in
population, land use development in the catchment
and inappropriate application of fertilizers in farms
were cited as the largest generators of nutrients
recorded in the Yarra River (Anonymous 2009).
Sources of nitrogen and phosphorus that find
their way into the Amala and Nyangores tributaries
are numerous and can range from livestock and
agricultural activities at the upper catchment areas
to urbanization and in-stream human activities like
use of detergents during washing of clothes and
utensils and bathing in the river as well as poor
human waste disposal. High protein intake in the
human diet is also regarded as a contributor of
nitrogen in water especially if the human waste is
poorly disposed (Anonymous 2002b); thus, this must
not be ruled out as a possible contributor of
nitrogen into the Mara River. While acknowledging
the increased role that anthropogenic activities play
in water quality degradation, a study by Liu et al.
(2008) singled out human wastes as the second
biggest source of nitrogen load into aquatic systems
after agricultural fertilizers. However, atmospheric
deposition is also an important source of nitrogen
into aquatic systems, although it is more
pronounced in lentic than lotic systems (Baron et al.
2009). Small-scale agricultural activities evident in
the upper Mara River catchment and along the river
channel in which fertilizers and manure are used are
also presumed to contribute to nutrient enrichment
of the Mara River waters. Likewise, livestock
activities along the riverbanks could also have
contributed to the nutrients as well as coliform
bacteria in the water through waste deposition
either along the banks or in the river channel.
Consistent with the current findings, previous
studies by Shindo et al. (2006) and more currently by
Matano et al. (2013) also found a correlation
between high levels of total nitrogen and
phosphorus with increased levels of E. coli along the
Mara River tributaries. They both attributed this to
land use types and wastewater discharged from
adjacent terrestrial ecosystems. It was highly
suspected that untreated wastewater and sewage
discharge from informal urban settlements and lack
of sufficient sanitation facilities could also have
contributed greatly to the high coliform levels
observed along the Mara River tributaries. This
proved to be the case when the relatively populated
and highly disturbed middle part of Bomet, along
the Nyangores tributary and exit point from Mulot,
along the Amala tributary, were found to contain
high levels of total phosphorus, total nitrogen and
total coliform bacteria, compared to the relatively
undisturbed upper catchment spring draining into
the Nyangores tributary.
These findings are consistent with those of
APHRC (Anonymous 2002c), in which the informal
settlements along the Nairobi River also
characterized by a high population and insufficient
sanitation facilities contributed considerably to its
water quality degradation. Bashir and Kawo (2004)
also showed a close link between effluent discharge
from highly populated informal settlements and
high nutrient levels and microbes in adjacent aquatic
systems, while WHO (Anonymous 2002b) reported
that lack of provision of septic tanks and sewage
systems can lead to surface water pollution. The
significant variation in total coliform levels observed
between sites along the two tributaries could,
therefore, be a result of localized small-scale
variability in water quality, driven largely by in-
stream anthropogenic and livestock activities at
specific points along the river continuum.
Escherichia coli, which are indicators of fecal
contamination in surface waters, varied significantly
between sites along the Nyangores tributary with
the highest levels recorded at points flowing
through the urbanized area (Bomet), while the
lowest were at an undisturbed upper catchment
spring that discharges its water into the Nyangores
tributary. This provides further evidence of the
7
Anyona et al.EurAsian Journal of BioSciences 8: 1-11 (2014)
8. Anyona et al.
8
EurAsian Journal of BioSciences 8: 1-11 (2014)
contributory role that anthropogenic and livestock
activities play in water quality degradation. A
previous study by Frenzel and Couvillion (2002) also
established a significant positive correlation
between fecal bacteria and high human population
density in urban watersheds. Okoko et al. (2012)
attributed increased levels of E. coli at some points
along the River Awach in Kisumu County, western
Kenya to localized contributions through
anthropogenic activities. Livestock grazing along the
river and use of certain sections along the river as
their watering points can also contribute to E. coli
bacterial loading in the river water either directly
through deposition of animal waste into the river, or
indirectly when animal waste deposited on land or
spread manure on agricultural fields surrounding the
aquatic system is swept into surface water bodies by
surface runoff. Since E. coli bacteria from humans
and other warm-blooded animals tend to die rapidly
outside the body, their presence in Mara River water
is a clear indication of a localized and more recent
contamination and, thus, a greater risk factor to
inhabitants of the Mara River Basin who depend on
the water for domestic use.
The distribution of E. coli along the Nyangores
and Amala tributaries was not uniform nor did it
increase or decrease exponentially downstream.
Instead, E. coli levels varied highly with more
disturbed areas recording relatively higher levels
compared to less disturbed areas. This was probably
due to the small-scale variability in water quality,
emanating from point source pollution that was
driven mainly by anthropogenic activities. Noble et
al. (2004) attributed the spatial variation in bacterial
levels to initial loading and the disappearance rate
which is a function of time and distance of travel
from the source, as well as other external and
internal factors such as temperature, competition,
toxicity, predation and solar radiation. This can to
some extent explain the spatial variability of
coliform bacteria observed at certain points along
the Amala and Nyangores tributaries.
The most significant finding in this study,
however, was the relatively high (27.5%) E. coli
proportions at the upper catchment spring draining
into the Amala tributary, previously thought to
contain clean water. The high E. coli levels at this
particular point of the spring should be of great
concern because of the critical role the spring plays
as the main source of water for domestic use by the
surrounding inhabitants. The US EPA (Anonymous
2012b) recreational water quality criteria put the
concentration threshold for culturable E. coli at a
mean of 126 cfu per 100 mL measured using EPA
Method 1603 or any other equivalent method that
measures culturable E. coli in a sample of water
meant for recreational purposes. However, the
thresholds are put at nil or 0 (zero) for waters meant
for human consumption. This implies that while the
Mara River waters can be used for recreational
purposes, they may not be safe for drinking.
Apart from human activities, livestock activities
coupled with the gently sloping and largely bare
landscape around this particular spring could be
facilitating the transfer of waste into the spring
contributing to high E. coli levels. Kelsey et al. (2004)
attributed fecal contaminants in surface water
systems to storm water runoff from urban and
agricultural land uses. Likewise, a longitudinal study
by Toothman et al. (2009) in North Carolina, in the
United States of America, singled out urban storm
water runoff as the major contributor to fecal
coliform bacterial loads in the surface waters.
As watershed areas are developed for residential,
commercial, industrial, and transportation land uses,
the quality and quantity of freshwater in rivers is
substantially altered due to increase in the
impervious layer. This is because many types of
pollutants, originating from a variety of sources,
accumulate over impervious urban surfaces and are
subsequently washed into water bodies during and
immediately following rainfall events, severely
degrading water quality and harming aquatic life.
This is however serious in areas like Olympia,
Washington where the impervious layer covering
over 63% has been recorded (Schueler 1994)
compared to the Mara River region where the
impervious layers are very minimal. For instance, a
recent land cover map of the Nyangores Basin
showed that the basin was covered by 64% cropland,
26% forest, 9% bushland, and 1% tea (Anonymous
2007b). Surface waters in such areas are likely to be
9. influenced more by agricultural pollutants and
sewage than urban surface runoffs.
Regression analysis showed that total nitrogen
was predictive of E. coli, along the two Mara River
tributaries. These findings are consistent with those
of Burkholder et al. (2007) and Mallin et al. (2000),
which reported significant relationships between
fecal bacteria and nutrient concentrations due to a
common source or a random arrival of nutrients and
E. coli in the same area. The detection of E. coli at
various sections of the Mara River tributaries was a
strong indication of the possible presence of other
potentially harmful enteric pathogens as was also
reported by Mireille et al. (2011) in their study on the
prevalence of pathogenic strains of E. coli in urban
streams in Cameroon.
The continued use of water from the Mara River
tributaries for domestic purposes without
knowledge of its sanitary quality, therefore,
continues to expose the Mara River Basin
inhabitants to an increased risk of contracting
waterborne diseases such as cholera, typhoid, and
hepatitis among others. It is, thus, necessary to
educate communities residing not only within the
Mara River Basin but also within other river basins
especially in developing countries, on basic hygienic
and sanitation practices aimed at curbing water
pollution and, therefore, reducing waterborne
diseases. In addition, all water collection points
including springs need to be protected to curb
backflows that may lead to source contamination,
while anthropogenic activities such as washing of
soiled baby clothing and bathing directly in the river,
or defecating along the banks should be
discouraged. Similarly, discharge of untreated
sewage into the river channel should also be curbed.
These recommendations are not only restricted to
the Mara River Basin, but can also be applied widely
to many other aquatic systems across the world that
are faced with similar issues.
We acknowledge the Lake Victoria Basin
Commission Secretariat for providing funds for this
study, Maseno University and Kenya Marine and
Fisheries Research Institute for providing the
required infrastructure and technical support during
sample collection and nutrient analysis and Longisa
District Hospital administration for use of their
laboratory for microbial analysis.
9
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Anonymous (2000) Measuring preferences on health system performance assessment. World Health Organization,
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