“ACTIVE BIOMONITORING OF TRACE

HEAVY METALS USING FISH

(OREOCHROMIS NILOTICUS) AS
BY: Z. Birungi, B.
BIOINDICATOR SPECIES. Masola, M.F.
Zaranyika, I. Naigaga,
B. Marshall
THE CASE OF NAKIVUBO WETLAND
ALONG LAKE VICTORIA”

BIOMONITORING
REVIEWED BY:
ASTRI YULIA
NIM: 1820942010 LECTURE:
DR.ENG.ZULKARNAINI
 ^ ^ ABSTRACT ^ ^
The Nakivubo wetland in Uganda, which feeds Lake Victoria at Murchison bay, has become
severely degraded over recent years and is particularly threatened by the expansion of
industry, settlement and cultivation on its fertile fringes. The aim of this study was to actively
biomonitor selected trace heavy metals using Nile tilapia (Oreochromis niloticus). Nile tilapia
was set in cages at six sampling sites in Murchison bay for a period of six weeks, and
sampling was done every 2 weeks for active biomonitoring and weekly for physico-chemical
variables. The control (site 7) was at the Aquaculture Research and Development centre,
Uganda. Fish tissue was dissected and gills, liver and muscle removed for heavy metal
analysis. Water samples and fish tissue from each site were analysed using Atomic
Absorption Spectrophotometer (AAS) to determine metal concentrations of zinc, copper,
chromium and manganese. Site 4 had the highest average NH4–N of 14.28 ± 12.73 mg/l
which was above effluent standards for Uganda. From the findings, there were significantly
higher concentrations of heavy metals in fish than in water (p > 0.01). Fish kills were
recorded highest for sites 4 (55%), 5 (77.5%) and 6 (60%) in less than six weeks indicating
high levels of pollution. Gills showed the highest accumulation of copper after a period of
six weeks as compared to other metals with a mean ± SE of 11.7 lg/mg wet weight. In
general, the order of accumulation of metals in tissue after six weeks was in order; copper >
zinc > chromium > manganese and gills > liver > muscle. In conclusion, active
biomonitoring was found to be a good tool for monitoring water quality as it integrates
responses to combinations of all contaminants there by indicating overall effects in a water
body. To improve the study, there is need to incorporate post mortem studies and
biomarker analysis since the later can give early warning of pollution before fish kills are
observed.
^ ^ INTRODUCTION ^ ^
• Heavy metals normally occurring in nature are
not harmful to the environment, because they
play an essential role in tissue metabolism and
growth of plants and animals (Amundsen et al.,
1997).
• However severe metal imbalances are toxic and
marginal imbalances contribute to deformities
and impede health. Such as silver, barium, cobalt,
manganese, molybdenum, sodium, thallium, iron
and zinc. These metals were selected because of
their potential for human exposure and increased
health risk.
• Nakivubo wetland in Uganda, which feeds Lake
Victoria at Murchison bay, has become severely
degraded over recent years, and is particularly
threatened by the expansion of industry,
settlement and small scale cultivation on its
fertile fringes (COWI/VKI, 1998). Currently there
are limited data available on heavy metal
pollution. The data available are mainly
restricted to types and quantities that pollute
the wetland in Kampala.
 The heavy metal
the animals
concentration

The heavy metal concentration in tissues reflects past exposure

via water and/or food and it can demonstrate the current
situation of the animals before toxicity affects the ecological
balance of populations in the aquatic environment (Forstner
and Wittmann, 1983)
^ ^ Research Purposes ^ ^
• Active biomonitoring involves the translocation of
organisms from one place to another and quantifying their
biochemical, physiological and or organismal responses for
the purpose of water quality monitoring (De Kock and
Kramer, 1994).

The specific objectives of this study were:

• (i) to determine the physical and chemical status of the
wetland at Murchison bay,
• (ii) to assess the suitability of Nile tilapia (Oreochromis
niloticus) as bioindicators for environmental pollution,
• (iii) to determine heavy metal accumulation in fish tissue
(gills, liver and muscle), and
• (iv) to compare the results of heavy metals in fish tissue
and water.
 ^ ^ Materials & Methods ^ ^
• Study Area
• Biomonitor : Nile tilapia (Oreochromis niloticus)
• Fish tissue was dissected and gills, liver and
muscle removed for heavy metal analysis
• Water samples and fish tissue from each site
were analysed using Atomic Absorption
Spectrophotometer (AAS) to determine metal
concentrations of zinc, copper, chromium and
manganese.
• Using 6 cages for research, and 1 cages for
control
• Period: in 6 weeks
 Sampling Protocol
The sample size was 40 fishes for each
cage with the reference site being the
fish farm. Sampling was done weekly
for physico-chemical parameters and
every two weeks for active
biomonitoring in a period of six weeks.

Juvenile Fish (Orechromosis Niloticus)

The first sampling for all the sites was

done after a period two weeks, during
which four fish and water samples
were taken from each site and
transported to the biochemistry
laboraories.
 STEP’s
Before dissection,
• each fish was weighed
and, total and standard
length were measured.
• The fishes that did not
survive field exposure
were thrown away
• Dissection was carried out on an aluminium foil
work place using stainless steel tools and wearing
surgical gloves. Muscle, gills and liver were
removed for metal analysis.
• After dissection, the tissue samples were wrapped
in aluminium foil and frozen at 80°C prior to metal
analysis.
 The Parameters in Research
Nu Parameters Instruments Methods
1. The Heavy Metals: Perkin–Elmer Manufacturer’s
copper, chromium, zinc 2380 Atomic Absorption manual (Hach
and manganese Spectrophotometer (AAS) Company
Procedures
2. The physico-chemical Manual,
variables : 1996–1999)
a. pH & temperature *WTW pH 330 meter, WTW GmbH and
Co., and described in
Weilheim, Germany detail in
b. DO *WTW Oxi 330 (Bartram and
Meter Ballance, 1996).
c. TDS *WTW LF 330 Meter

3. Others Parameters: a Hach water analysis kit and a Hach

turbidity, nitrate, DR/2010 portable data logging
nitrite, ammonia- spectrophotometer (Hach Co., Loveland,
nitrogen, calcium, Co., USA) using filtered water samples
potassium, sulphates (Whatman GF 47 mm filters).
and chlorine
 Preparation Analysis Methods
*For Preparation of fish *Food and Agricultural Organization
tissues,

*For Preparation of stock *A Shimadzu AA 6401F Atomic

standard solutions Absorption Spectrophotometer (ASS) was used to
determine
the levels of copper, manganese, zinc and chromium
in these tissues

STATISTIC ANALYSIS

The physico-chemical variables for water were analysed using Microsoft

excel to determine mean and standard deviation and graphs were plotted
with the aid of 2004 SYSTAT software (Sigma plot (9)). To do a univariate
analysis of only two groups (mean concentrations of trace heavy metals in
water and fish), the t-test was employed. Analysis of variance (ANOVA) was
used to analyse bioaccumulation of heavy metals in tissue.
RESULTS
Physical–
chemical
parameters of
water:

Electrical
conductivity was
highest in site 6
with an average
concentration of
222 ± 75.97
ls/cm.
Sites 3,4, and 5 had the
lowest average
dissolved oxygen levels
of
1.8 ± 1.02 mg/l, 1.55 ±
0.54 mg/l and 1.23 ±
0.06 mg/l,
respectively (Fig. 3b).
Site 4 had the
highest average
NH4–N of 14.28
± 12.73 mg/l
which was
significantly
different from
other sites.
• The pH range and
temperature for all
the seven sites was
within permissible
effluent standards
for Uganda 6–8 and
20–35 C,
respectively. (see
Figs. 3c and 3e).
 Nile Tilapia as bioindicators of water pollution

Forty Nile tilapia fishes were stocked in cages in different sites

at Murchison bay, which was highly polluted from domestic and
industrial effluent. Site 5 had the highest number of fish kills
(77.5%), followed by Site 6 (60%) and Site 4 (55%). Of the fish
stocked in Site 7 (control), 100% survived (Table 1). In general,
Sites 1, 2, 3, 4 and 6 still had some fish surviving by the fourth
week as shown in Table 1.
Comparison of heavy metals in water and
Nile Tilapia
• A preliminary test was carried out to test the
hypothesis of concentration of heavy metals in
water significantly different from those in fish
using t-test in Genstat.
• Results from this analysis showed that the
mean amount of all the tested metals in fish
was greater than that in water (p < 0.001).
Comparison of heavy metals in fish tissue, sites
and period
After carrying out
ANOVA, the results
for copper
showed significant
differences in tissue
and period (p < 0.05)

Site 1 showed the highest

accumulation of copper after
a period of 2 weeks in liver
(20 lg/mg wet weight) but
which reduced to 2 lg/mg
after 6 weeks
 There was
bioaccumulation of
copper in
muscle for all the
sites except 4, 5 and
6 which had no fish
left by the sixth
week.

There was bioaccumulation of copper in muscle after

a period of 6 weeks except for Sites 4, 5 and 6 which
had no fish left by the sixth week.
 The results for
manganese showed
significant differences
in tissue, site and
period (p < 0.05) as
shown in Table 2.
Site 2 had the highest
bioaccumulation of Mn in liver
with an average concentration of 4
lg/mg after a period of 6 weeks

Site 3 showed- with an average

the highest concentration of
bioaccumulation 2 lg/mg wet
after a period of- weight.
6 weeks in gills-
Gills showed the
highest
accumulation of
copper after a
period of six weeks
as compared to
other metals with
a mean ± SE of
11.7 lg/mg wet
weight. In general,
the order of
accumulation of
metals in tissue
after 6 weeks was
copper > zinc >
chromium >
manganese and
gills > liver >
muscle.
 The ANOVA results for
zinc showed significant
differences
in tissue p < 0.05
(Table 2).

Site 3 had the highest bioaccumulation

in gills after a period of 6 weeks with an
average concentration of 10 lg/mg (Fig. 6c).

Site 6 also showed bioaccumulation in

muscle after a period of 4 weeks with
an average of 6 lg/mg wet weight (Fig.
6b).
The results for chromium showed
significant differences in tissue and
period, p < 0.05 (Table 2). Although
in general chromium recorded the
lowest levels in Oreochromis niloticus
as compared to other heavy metals,
there was bioaccumulation
throughout the period of 6 weeks
(Table 3).
• Site 4 had the highest average NH4–N of 14.28 ± 12.73
mg/l which was above effluent standards for Uganda.
From the findings, there were significantly higher
concentrations of heavy metals in fish than in water (p
> 0.01). Fish kills were recorded highest for sites 4
(55%), 5 (77.5%) and 6 (60%) in less than six weeks
indicating high levels of pollution.
• Gills showed the highest accumulation of copper after
a period of six weeks as compared to other metals with
a mean ± SE of 11.7 lg/mg wet weight. In general, the
order of accumulation of metals in tissue after six
weeks was in order; copper > zinc > chromium >
manganese and gills > liver > muscle.
 Conclusion
• active biomonitoring was found to be a good
tool for monitoring water quality as it
integrates responses to combinations of all
contaminants thereby indicating overall
effects in a water body. To improve the study,
there is need to incorporate post mortem
studies and biomarker analysis since the later
can give early warning of pollution before fish
kills are observed.