J. Int. Environmental Application & Science, Vol.

9 (2): 284-292 (2014)

Mosquito fish, Gambusia affinis (Baird & Girard, 1853) as Bioindicator for
Water Pollution with Lead
Adel Mashaan Rabee*, Wasan Qasim Turki

Department of Biology, College of Science, Baghdad University, Baghdad, Iraq,

Received March 20, 2014; Accepted June 02, 2014

Abstract: Acute and sub-chronic tests with lead were conducted on mosquitofish
(Gambusia affinis) by determining LC50 values and their 95 confidence interval end points
for 24,48,72 and 96 hr exposure. The LC50 were estimated as 9.6, 6.95, 4 and 3 mg/L for 24,
28, 72 and 96hr respectively. The behavioural response observed in the fish were erratic
swimming, loss of reflex, hyperactivities and hyperventilation. These effects increased with
increasing concentration of the lead and duration of exposure. The concentration factor for
lead in the Gambusia affinis ranged between 0.0093 and 0.022. The histological
examination of the gills of the fish after acute and subchronic exposure (34 days) showed
pathological changes and alterations such as epithelial necrosis, hypertrophy of the
epithelial cells and epithelial separation. This study investigated the occurrence of
resistance to lead in the Mosquitofish, and the results showed elevated lead resistance in
testing fishes as a result of acclimation (physiological) after were kept for 34 days under
safe concentration of lead.
Keywords: Gambusia affinis, LC50, Resistance to lead, Responses, Bioindicator

Introduction
Natural and anthropogenic sources continuously release heavy metals into aquatic ecosystems. The
heavy metals after reaching to freshwaters cause serious problem due to their long persistence,
bioaccumulation in the food chain, and toxic to the organisms. Fish, being dominant inhibitors of the aquatic
environment, are considered as indicators for heavy metal pollution (Srivastav et al., 2013). When fishes are
exposed to high level of metal ions in aquatic environment, their tissues tend to take up these metal ions
through various routes from their surroundings. There are two main routes of metal acquisition; directly from
the water and from the diet (Bury et al., 2003). Certain contaminants, particularly some elements and organic
compounds, may be accumulated in the tissues of organisms. Therefore, chemical analysis of the appropriate
biological tissues can be used to show that the organism has been exposed to contaminants and, in some
cases, to monitor the spatial distribution, or accumulation, of that contaminant in the aquatic ecosystem
(Plafkin, 1989). As highly visible and valuable components of the freshwater ecosystems, fish communities
have been applied to monitor the river ecosystem health for a long time (Fausch et al., 1990 ; Simon, 1991).
Fish have been used extensively in biological early warning systems (also referred to as biomonitors) rely on
their excellent response to different contaminates in water (Bartram & Balance, 1996). Bio-monitor
organisms can be used to estimate bioavailable contaminant concentrations provided that a reliable model
exists between organism and environmental contaminant concentrations (Croteau et al. 1998). Mosquitofish,
Gambusia affinis (Baird & Girard,1853) is the most common fish in the various Canals in Iraq. In the present
study, we aimed to provide information about the following points:-
1. To investigate the toxicity and occurrence of resistance to lead in the G. affinis .
2. To examine the possibility of using G. affinis as potential biomonitor of water pollution by lead as a part
of continuous bio- monitoring programs and biological early warning systems.

Materials and Methods

Experimental animals
Fish G. affinis (with average length 3cm) were obtained from AL-Jadyria Canal which connected with
Tigris River in Baghdad city. The fishes were acclimatized for one week in glasses aquaria(80 × 30 × 30 cm)

*
Corresponding: E-Mail: adilrabee@yahoo.com; Tel: +9647901209570
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prior to the toxicity test. The fish were not fed prior to the test in order to minimize the production of waste
thereby reducing ammonia production from the wastes.

Water properties
Determination of lead concentration in fish specimens, AL-Jadyria Canal, Tigris River and filtered water
was done by Flameless Atomic Absorption Spectrophotometer (Shimadzu- GFA- 413 Graphite Furnace
Atomizer, Japan). The pH, conductivity and total dissolved solids were conducted during the experiment
using a portable pH/ EC/ TDS meters. Dissolved oxygen was measured by Winkler method. Also water
quality monitoring was done prior to the experiments, during the experiments and after the experiments.

Determination of LC50
For determination of LC50 , six varying concentrations of lead (1, 2.5, 5, 10, 20 and 40 mg/L) were
prepared. These varying concentrations were prepared arithmetically. Lead nitrate dissolved in deionized
water, and then the desired volume of the solution was mixed in freshwater to obtain the above-mentioned
lead concentrations. Eight fish were introduced into each aquarium for acute tests. The first aquarium
contained fishes living in non treated freshwater and considered as controls. The numbers of dead fish were
counted daily and removed immediately from the aquaria. Behavior of tested fish in all tested concentrations
were monitored.

Subchronic experiments
Safe concentration (Sc) of lead was determined following the formula given by Hart et al., (1945):
Sc = LC50 (24hr.) – 0.3/[LC50 (48hr.)- LC50(24h.)]x
Where, safety factor equal 2 or 3

For the sub-chronic experiment, the mosquitofish (G. affinis) (after one week acclimatization) subjected
to 0.5, 1 and 1.5 mg/L for 34 days. Concurrently, a control group was also run. The media (both control and
experimental) changed every 48 h. After sub- chronic exposure to lead fish again subjected to LC50 test by
using same concentrations as mentioned above to determine the resistance to lead in tested fish. Water of
each aquarium was changed on every 5th day and Pb(NO3)2 was maintained throughout the experiment
duration of 34 days.

Bio-concentration factor
Bio-concentration factor (BCF) of lead in testing fish was determined according to the formula:
BCF=CB/CW
Where, CB = the concentration of the chemical in an organism
CW= the concentration of chemical in water

Histological changes
Furthermore, after exposure for different concentrations of lead nitrate, both treated and untreated fish
were sacrificed by giving a sharp blow on the head and dissect out gill were removed and washed in saline
water to remove blood and fixed aqueous fixative for 24hrs. The fixed samples were dehydrated in ascending
series of ethanol, cleared in methyl benzoate and embedded in paraffin wax. Sections of 6 microns thickness
were cut out, mounted and stained with Hematoxylin and Eosin for examination by light microscope.

Statistical analyses
All experiments were repeated three times and performed in triplicate. Data were analysed with SPSS
statistical analysis software (Version 13.0). The LC50 values were calculated using Probit analysis statistical
method. The MS Excel 2007 was used to find regression equation (Y= mortality percentage; X=log of
concentrations), the LC50 was derived from the best-fit line obtained. Differences among the results were
considered to be statistically significant when P value was < 0.05. Also, standard error (SE) (with 95%
confidence limits) for LC50 can be calculated according to the formula was mentioned by Ghosh (1984).

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Results and Discussion

Water properties
During the collection period of G.affinis some physico-chemical properties and lead concentration in
water from AL- Jadyria Canal and the aquaria were determined. Tables 1 and 2 shows the values of these
parameters. Overall, all the studied parameters and concentration of lead were within suitable limits for fish
life. Gambusia sp. have a remarkable ability to withstand adverse conditions (McKay et al., 2001). The
species is extremely tolerant of poor water quality in particular high turbidity, extremes of temperature and
salinity ranges, and low dissolved oxygen (Karolak, 2006).

Table 1. Physico-chemical parameters and concentration of lead in water from AL- Jadyria Canal during the
year 2013
Parameter Range Mean & SD Optimum value for aquatic
life*
Temperature (oC) 18 – 20 19±1 10-32
Dissolved Oxygen (mg/L) 6.6 – 7 6.8±0.2 ˃5
Hydrogen ion concentration 7.35 – 8.1 7.7±0.4 6.5-9
TDS (mg/L) 350-370 360±10 <400
Electrical conductivity( µS/cm) 620-660 640±20.8 <1800
Lead (mg/L) 0.01-0.15 0.05±0.039 <0.65
*Optimum values taken from Lumb et al., (2006) and (USEPA, 2002) , SD =Standard deviation

Table 2. The physico-chemical parameters and concentration of lead in the aquarium water
Physical and chemical properties Range Mean and standard
deviation
o
Temperature ( C) 18-22 20±2
Dissolved Oxygen (mg/L) 6.5-7 6.75±0.26
Hydrogen ion concentration (pH) 7.5-7.9 7.7±0.20
TDS (mg/L) 380-410 395±15
Electrical conductivity( µS/cm) 600-630 610±17
Lead (mg/L) 0.001-0.003 0.002±0.001

Behavioural responses
After acute experiments, all fish exposed to lead (except fish exposed to first concentration) showed
hyperventilation and rapid gill movement, inclined swimming and loss of reflex (Table 3). As the duration of
the experiment increased the tested fish showed increased in weakness, motionless and curvature in the
middle of the fish’s body. Changes in behavioral patterns exhibited by fish were possible to counteract
aquatic hypoxia condition possibly caused by the chemicals (Kind et al., 2002). Hiatt et al. (1993) stated that
respiratory poisons induce symptoms of gulping, swimming at the surface, and depressed activity.
Biomonitoring should rely on sublethal endpoints rather than mortality alone. Behaviour is a sublethal
parameter that is readily altered by stress.

Table 3. Behavioral responses of Gambusia affinis during acute exposure to lead (- = absent ; * = present)
Concentrations mg/L
Behaviour Control 1 2.5 5 10 20 40
Rapid gill movement - - * * * * *
Inclined swimming - - * * * * *
Loss of reflex - - * * * * *
Hyperventilation - - * * * * *
Emotion nervous - - - - - - -

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Median Lethal Concentration (LC50)

From the results of mortality after to exposed to lead, LC50 values were calculated to be 9.6, 6.95, 4 and 3
mg/L for 24, 48, 72 and 96hr. respectively (Figures 1,2,3 and 4, Table 4) . Compared with other fishes G.
affinis considered very sensitive for lead. For explanation, Nekoubin et al. (2012) found that the median
lethal concentration (LC50) of lead for grass carp (Ctenopharyngodon idella) after exposed to 24, 48 and 72
hr were 293, 278 and 260 mg/L respectively. Abedi et al. (2012) studied the LC50 of lead nitrate into the
Common Carp (Cyprinus carpio) and they found that the LC50 was 77.33 mg/L. During acute exposure,
waterborne Pb can cause disruption of Ca2+, chlorides, sodium, spinal deformities, black tail disease and
disruption in the synthesis of hemoglobin in fish (Dallinger et al., 2002). Also, the results showed that the
values of LC50 were found to be 2.4mg/L in males (Figure 5), while in females was 4.3 mg/L (Figure 6)
during 96hr of exposure. Sexually dimorphic nature of G. affinis may cause the differential mortality rates
(Meffe & Snelson, 1989). Male of G. affinis are smaller and more sensitive to environmental stress
(Krumholz, 1944).

Table 4. LC50 values of lead (mg/L) and their 95% Confidence Limits
Time (hours) LC50 (mg/L) ± 95 % Confidence
Standard Error Limits
24 hr 9.6 ± 3.9 5.7-13.5
48 hr 6.95± 4 2.9-10.95
72 hr 4 ± 2.5 1.5 - 6.5
96 hr 3 ± 1.4 1.6 - 4.4
96hr for female 4.3 ± 2.7 1.6-7
96 hr for male 2.4 ± 1.7 0.7- 4.1
24 hr after sub-chronic exposure 16.2 ± 8.5 7.7-24.7

Figure 1.Toxicity curve of lead to the G. affinis after Figure 2.Toxicity curve of lead to the G. affinis after
24hr. of exposure 48hr. of exposure

Figure 3. Toxicity curve of lead to the G. affinis after Figure 4. Toxicity curve of lead to the G. affinis after
72hr. of exposure 96hr. of exposure

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Figure 5.Toxicity curve of lead to the male of G. Figure 6.Toxicity curve of lead to the female of G. affinis
affinis after 96hr after 96hr

Figure 7. Toxicity curve of lead to G. affinis after sub-chronic exposure

Subchronic exposure
Three series of exposure were conducted after we calculated safe concentrations for long-term exposure
to lead (Table 5). In series 1, fish specimens exposed to 0.5 mg/L of lead, while in other series experiments
studied fish placed in 1 and 1.5 mg/L respectively. In this exposure, no mortality was observed at all periods
of exposure for each tested concentrations. Also, fish growth was normal and did not differ from the growth
that was happening in the control aquarium, the same applies to the activities of swimming except in the
highest concentration(1.5 mg/L) we observed slow in fish movement. No effect of lead in following
concentrations (0.5 and 1 mg/L) on feeding behaviour but in 1.5 mg/L, the studied fish reduced from their
diet. Movement of gills was normal and similar to their counterpart's fish control.

Table 5. Safe concentrations values of exposure to lead

Element Sc, when x=2 Sc, when x=3
Lead 0.7 1.5

Resistances to lead in Gambusia affinis

After long-term exposure to lead took fish from highest concentration of lead 1.5 mg/L and subjected to
the acute test and calculated LC50 by using the following concentrations 1, 2.5, 5, 10, 20, and 40 mg/L. The
results indicated a significant increase in the value of 24hr LC50 (16.2 mg/L) (Figure 7,Table 4) compared
with LC50 before chronic exposure. This metal regulation is due to the induction of low molecular weight of
metal-binding proteins, such as metallothionein, which are closely related to heavy metal exposure, and
metals taken up from the environment can be detoxified by the liver (Roesijadi & Robinson, 1994; Kotze,
1997). During acclimatization for heavy metal, this protein, (metallothioneins) are released in the body of the
organism and detoxify the metal ions (Deb & Fukushima, 1999). This may cause higher concentrations of
heavy metals being required to cause effects and lead to high values of LC50.
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Bio-concentration factor (BCF)

Metals can enter the fish through gills, food particles or/and skin after ingestion, metals would start
accumulation in the body organs depending upon the intensity of their uptake (Viondhini & Narayanan,
2008). Results of bio-concentration factor (BCF) conducted after sub-chronic exposure, indicated that the
lowest value recorded at the highest concentration, while the high value associated with the lowest
concentration in aquarium water. The weaker accumulation in the present work may be returned to found the
efficiency mechanisms of detoxification which observed in G. affinis (according to resistance experiment).
Klerks & Lentz (1998) had exhibited an increased resistance to lead in the pollution region and accumulate
this metal in the body of tested fish compared with reference region. The result of the present work may be
due to the period of exposure is relatively short as well as interference of other environmental factors. The
accumulation of metals in fish tissues is dependent upon exposure concentration, duration and other factors
such as salinity, temperature, hardness, metabolism of the animals, biological and genetic factors and species
age (Kotze et al., 1999; Otitoloju, 2001; Karthikeyan et al., 2007; Bu-Olayan & Thomas, 2008;
Kamaruzzaman et al., 2010). Mohanambal & Puvaneswari (2013) referred that the bioaccumulation of lead
in the tissues of organisms was increased with duration of exposure periods.

Table 6. Bio-concentration factor of lead for Gambusia affinis after sub-chronic experiments
Lead concentration in Lead concentration BCF
aquarium water(mg/L) in fish (mg/L)
0.5 0.011 0.022
1 0.012 0.012
1.5 0.014 0.0093

Histological changes
Histological examination of G. affinis gave significant indication of lead. The effects include, gill
alteration such as changes in secondary lamellae (SL) which converted to clumping shape (CL Shape), fusion
of secondary lamellae and degeneration of lamellar epithelium and epithelial necrosis, hypertrophy of the
epithelial cells, epithelial separation (ES), loose in regular shape of epithelium(LR) and rupture of gill
hemorrhage at primary lamellae(PL) (Figures 9,10,11and 12), while there was no pathological lesion in the
control gills (Figure 8). The histopathological changes caused by different chemicals in gills and other organs
of fishes were reported by Banerjee and Chandra (2005) and OIojo et al. (2005). In the present study, the
gills showed severe degeneration extend to adjacent primary and secondary lamella. Martinz et al. (2004)
stated that heavy metals were associated with lamellar aneurism lesion that seems to involve pillar cell
disruption. Overall, these lesions are nearly similar to the structural changes, which have been described by
many investigators (Clark et al.,1997; Mazon et al., 2002; Banerjee & Chandra, 2005).

Figure 8. Gills of G. affinis in the control aquarium shows no pathological in primary

lamellae(PL) and secondary lamellae (SL). ×100

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Figure 9. Gill of G. affinis exposed to 40 mg/L of Figure 10. Gill of G. affinis exposed to 0.5 mg/L of
lead shows primary lamellae (PL) and lead shows Loose in regular shape of
secondary lamellae(SL). ×400 epithelium(LR). × 400

Figure 11. Gill of G. affinis exposed to 0.5mg/L of lead Figure 12. Gill of G. affinis exposed to 1.5 mg/ L of
shows epithelial separation (ES) lead shows clumping shape (CL). × 250

Conclusions
Our findings indicate that the toxicity of lead to the G. affinis were much higher when it compared with
other fishes, and males of G. affinis were more sensitive to the lead toxicity than females. Study of adaptation
in G. affinis showed the presence of effective resistance to lead toxicity and this case confirmed by high
value of LC50 calculated after sub-chronic exposure test. Nonetheless, the results of histological changes in
gills and behavioural response followed exposure to lead, demonstrated that it is possible to use G. affinis as
bio-indicators in the biomonitoring programs and the detection of water pollution with lead .

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