Introduction
The investigation of the accumulation of heavy metals in aquatic organisms specifically has been of growing interest over the last few decades. It has been established that heavy metals are severe in their action due to their persistence biological amplification through the food chain (Shah and Altindau, 2005) and they are not usually eliminated either by biodegradation or by chemicals means, in contrast to most organic pollutants (Ashraf et al., 2012). Heavy metals are natural components of the earth crust and are found in natural waters through the run-offs from washed soils and rocks. Human has continued to be the main cause of heavy metal introduction in the environment through the release of partially-treated or non-treated wastes from its various domestic and industrial activities (Olagunju, 2015).

Aquatic environments are usually the ultimate destination of pollutants including the land-based derived pollutants. Therefore, anthropogenic activities threaten the quality of the aquatic environments including the living and non-living resources present in them. The devastating effects of heavy metals on the ecological equilibrium and the diversity of aquatic organisms have been widely reported (Farombi, et al., 2007; Olagunju, 2015). Heavy metal at a particular concentration is capable of disrupting the normal physiological function of aquatic lives (Dahunsi et al., 2012;Ere et al., 2014). Fishes are permanent dwellers of water bodies and are therefore unavoidably exposed to the detrimental effects of these impinging pollutants. Clarias gariepinus have been widely used to evaluate the health status of aquatic ecosystems and also to assess the potential effects of heavy metals (Atolaye et al., 2006; Osasona et al., 2011; Opaluwa et al., 2012) which could be use to predict possible effects on other organisms including man.

Accumulation of heavy metals and its rate could be influenced by several factors. Such factors could be extrinsic (such as proximity to pollution source, quantity and rate of introduced pollutants etc.) or intrinsic (such as age, size, weight, sex, reproductive condition and a limiting metal retaining ability). There is the need to identify and understand the factors responsible for some organisms to accumulate more heavy metal than others. Thus, this research aims to investigate how exposure period and body size affects the level of bioconcentration of heavy metals in Clarias gariepinus.

Materials and Methods
The Clarias gariepinus (African catfish) used for the study was collected from the Ministry of Agriculture, Fisheries Division, Ogbomoso, Nigeria. For the exposure-period-dependent test, juvenile fish were used, but for the size-dependent test, blocking was first done to group the fish based on their body size (length). In each block, 5 individual fishes were randomly picked to make the experimental units. In each test was control group of 5 fishes. Prior to 7 days of acclimatization, the fish were starved for 24 h. The 12 h photoperiod was observed throughout the experimental study and the fish were fed once in a day with commercial feed pellet. Analytical graded cadmium chloride [CdCl₂], lead nitrate [Pb (NO₃)₂], potassium dichromate [K₂Cr₂O₇] and hydrated copper sulphate [CuSO_4.5H₂O] were used as metal toxicants.

Exposure-period-dependent test: Thirty (30) juveniles of the fish (15.6 ± 0.2 g; 12.1± 0.5 cm) were exposed to a pre-determined sublethal concentration of 5 mg/l of the combined metal solution for 21 days. The solution was renewed every 3 days. Mean value of accumulated heavy metal were periodically assessed every 3 days from 3 randomly picked fishes. The obtained metal concentrations (µg/g dwt.) were plotted against the days of exposure.

Body-size-dependent test: Five experimental units with 5 individual fishes in each were formed as follows: A (15.6 ± 0.2 g; 12.1± 0.5 cm); B (50 ± 5.0 g; 15 ± 2.6 cm); C (150 ± 15 g; 22 ± 4.2 cm); D (300 ± 20 g; 28± 5.1 cm) and E (500 ± 35g; 40 ± 5.7 cm). Each group was exposed to sublethal concentration of 5mg/l of the combined metal solutions for 21 days. The solution was renewed every 3 days. The mean values of the heavy metals were assessed at the end of 21 days exposure from 3 randomly picked fishes from each unit. The obtained metal concentrations (µg/g dwt.) were plotted against each standard length. Regression lines were fitted by the method of least squares.

Digestion and Analysis of Heavy Metals: Fish from each group were sacrificed. The body tissue was removed and oven dried at 70 - 73℃until a constant weight was obtained. The specimens were then grinded to fine powder and stored in desiccators in order to avoid moisture accumulation before digestion. FAO/SIDA (1983) procedure of digestion was adopted. This involved adding freshly prepared mixture of HNO₃/H₂O₂ (1:1) (15 ml) to each sample portion, stirred and covered with wash glass to allow the initial effervescence to subside. Then, the reaction mixture was heated slowly with continuous stirring for 20 minutes in a fume cupboard. After digestion, the beakers and its contents were then allowed to cool; filtered and distilled water was added to the 100ml mark of the volumetric flask. The heavy metals were then analyzed using Atomic Absorption Spectropho- tometer. The obtained data were statistically analyzed with SPSS 10 computer statistical software package by using one way analysis of variance (ANOVA) followed by Duncan Multiple Range Tests as a post-hoc test.

Results
The results of exposure-period-dependent test indicate that there was an initial increase in the concentrations of lead (Pb), cadmium (Cd), copper (Cu) and chromium (Cr) in the fish body tissues up to about the 12 th day of the exposure period followed by a gradual uniform or constant metal concentrations as exposure period increases (figure 1). In the experimental groups, the range of Pb was 1.38 ± 0.26 – 7.83 ± 0.48, Cd was 0.30 ± 0.18 - 1.91 ± 0.75, Cu was 1.35 ± 0.22 - 6.77 ± 0.34 and Cr was 1.29 ± 0.51 - 4.08 ± 0.25 within the 21 day of exposure period.

Figures 2 to 5 shows that the concentrations of Pb, Cd, Cu and Cr respectively in the body tissues of the fish decrease with increasing body size. The heavy metal concentrations (µg/g dwt.) recorded in the samples from the smallest body sized fish to the largest ranges as follows: Pb was 7.83 ± 0.48 - 1.38 ± 0.26, Cd was 1.91 ± 0.75 – 0.32 ± 0.20, Cu was 6.768 ± 0.34 – 3.01 ± 0.22 and Cr was 4.08 ± 0.26 – 2.27 ± 0.31. In this study, the order of heavy metal accumulation in the tissue of the fish was Pb>Cu>Cr>Cd.

Discussion
The use of biological assay that is easy, cheap and strongly related to the level of pollutants in the environment is desirable. However, bioassays are influenced by several extrinsic and intrinsic factors which cause a wide variability of results. Hence, it is important to put such factors such as body size and exposure period into consideration when biological assay is carried out to allow better interpretation and understanding of biological effects of a pollutant. Relationship exists between the exposure period to a pollutant and metal concentration in fish tissues (Jezierska and Witeska, 2006; Vinodhini and Narayanan, 2008; Dahunsi et al., 2012) and also between the age, size of fish and the concentration of metal in their tissue (Scerbo et al., 2005; Damodharan and Reddy, 2013).

Accumulation of metals from the environment is a function of uptake and elimination rate. The increase in the lead (Pb), cadmium (Cd), copper (Cu) and chromium (Cr) concentrations at the initial exposure period is in close agreement with the findings of Jezierska and Witeska (2006); and Vinodhini and Narayanan (2008) that, there is positive correlation between the exposure period and heavy metal concentration in the exposed fish. The uniform/constant metal concentration that was observed after the initial increase is in agreement with the study of Murugan et al. (2008) that the concentration of a metal becomes uniform or remains constant and later decreases as exposure to a sublethal concentration of a toxicant is prolonged. This uniformity may be indicative of the induction of regulatory processes in order to maintain the health of the fish and ensure its survival. Regulatory processes ensures the adaptation of the fish to the new condition and may involve establishment of an equilibrium between the rates metal uptake and rates of its excretion (Jezierska and Witeska, 2006) or involve the synthesis of polyphenols which are strong chelators of heavy metals in solution (Topcuoglu et al., 2003; Stengel 2006).

Bioactive metals are important in metabolism, thus in physiology and pathology of fish. The inverse relationship between the metal concentration and the body size/length of the fish is in consonance with the reports of De Wet et al. (1994). Allen-Gill and Martynov (1995) also observed an inverse correlation between the age (body size) and lead content in Coregonus clupeaformis. Balaji and Rao (2000) in their study find out decreased concentrations of copper, zinc, lead and cadmium with increasing shell length in Mytilopsis sallei. This is also supported by the report of Canli and Atli (2003) that youngest fish showed the highest concentrations of chromium, lead and copper. This may be due to higher metabolic activities in the younger fish compared to the older ones. The highest concentration of Pb may be due to its high selective affinity with fish tissue compared to the other metals. Cu functions as a cofactor in several enzyme systems but however, when in excessively high concentrations, it may pose serious threats to normal metabolic processes (Safahieh et al., 2011). Cr and Cd are non-essential and have no known biological benefit, but are rather detrimental to fish and human even at very low concentration. Cd had the lowest concentration but has been reported to be one of the most toxic heavy metals (Dural et al., 2006; Yilmaz et al., 2007) as it has high retention within the organism body where it may cause a lingering toxicity.

Conclusion
In this study, the concentrations of lead, cadmium, copper and chromium increase with the exposure period up to a particular point followed by uniform or constant metal levels. Negative correlation was recorded between the metal concentration in the fish and the body size/length of the fish. Hence, consideration of exposure period and size in relation to heavy metal concentration in fish is necessary for a better understanding and reporting of the effects of the metals.

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