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) *
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J. Int. Environmental Application & Science, Vol. 9 (2): 284-292 (2014) 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). 285
J. Int. Environmental Application & Science, Vol. 9 (2): 284-292 (2014)
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
