Bioaccumulation of Hexavalent Chromium in Tissues of a Freshwater Fish

This study aims to investigate the bioaccumulation Tissues. The experiment was carried on for 28 days, meanwhile sampling fish weekly. With continued exposure, the accumulations were increased and fish progressively lost their ability to respond to this increase in exposure period. The control group displayed normal behavior during the test period. The lowest concentrations (10 and 15 mg/L Cr6+) had similar behavior to that of the control group. From the dose 20 mg/ L Cr6+, the fish started to show behavioral disorders. 25 mg/L Cr6+ and at the highest concentration (30 mg/L Cr6+) onwards, there was shivering, rather high respiratory disorder and swimming in capsized position in the following order the concentrations of Cr in gills were almost twice, three and four times as high as in the muscle at 20, 25 and 30 ug/L Cr6+ mediums, respectively. The Cr accumulation on the skin of Tilapia for all experimental concentrations was nearly twice as much as in the muscle of samples at the end of experiment. Bioaccumulation of Hexavalent Chromium in Tissues of a Freshwater Fish Hussien M El-Shafei*


Introduction
The overall toxic impact on organs like gill, kidney and liver may seriously affect the metabolic, physiologic activities and could impair the growth and behavior of fish [1]. Acute poisoning by chromium compounds causes excess mucous secretion, damage in the gill respiratory epithelium and the fish may die with symptoms of suffocation [2]. On chronic exposures, hexavalent chromium severely affected the renal tubules causing hypertrophy of epithelial cells, reduction of tubular lumen [3], Chromium compounds also cause renal failure leading to the loss of osmoregulatory ability and respiration in fish [4]. Sub lethal effects of chromium in fish were directly related to the inhibition of various metabolic processes [5]. The hexavalent chromium induced depletion in the profiles of liver glycogen, total protein and total lipid has been reported [6,7] studied African catfish (Clarias gariepinus). Concentrations used varied from 11 to 114 mg/L (K 2 Cr 2 O 7 ). They found that the main deformity reported was abnormal body axis [8]. After 45 days of exposure significant decline in the protein and carbohydrate content of gills was observed [9]. The objective of our study was to investigate the impact of different chromium (10,15,20,25 and 30 ug/L Cr 6+ ) pollutions on the survival of juvenile Oreochromis aureus (Tilapia) and to compare the chromium concentrations in fish tissue (muscle, skin and gill).

Fish
O. aureus (Tilapia), a tropical freshwater and important culture fish, is a species commonly found in brackish water in estuaries all over the world [10][11][12]. Sixty Juvenile specimens of Tilapia were captured from ponds at Manzalla Lake and transferred to the laboratory where the experiments were conducted during April 2015. The samples were placed in aquarium for one week to allow for adaptation of the fish to the new conditions. Tap water used for the experiment had a pH value of 7.5 ± 0.3 and total hardness of 131 ± 5.3 mg CaCO 3 /L. The aquariums were aerated with air stones for proper oxygen saturation (8.5 mg O 2 /L). Fish were hand-fed once a day with artificial feed meal with commercial dry pellets. The feed was comprised of 32% crude protein, 22% crude lipid, 25% ash and 12% moisture. Fish were fed at a feeding rate of 3% of body weight per day for 28 days. Any fish that showed abnormal behavior were removed immediately from the tanks. Acclimatized fish were moved at random into six aquaria volume (50 L), five aquaria for each concentration and one aquarium for control, each containing ten fish.

Chemicals
All reagents were analytical grade. Required is (Merck). A Cr (VI) stock solution was delivered to five test aquariums via automatic pipettes. (Cr 6+ as 10, 15, 20, 25 and 30 ug/L)

Experimental exposure
Treatment water was monitored every day for dissolved oxygen, pH and Cr concentrations. Exposure period was 28 days, during which samples were taken on day 7, 14, 21 and 28. Experimental units were checked daily for mortality and behavioral changes and any dead fish was immediately removed from the aquariums. For measurement purpose, 3 fishes were taken from each aquarium and their lengths were measured (cm) and weighed (g) before dissection with cleansed tools.

Water and fish tissue analysis
Samples of 100 ml of water from each treatment were filtered through a 0.45 μm micropore membrane filter and stored at -20°C prior to analysis the chromium concentration in water (Cr 6+ mg/L). Each filtered sample bottle acidified to 1% HNO 3 and analyzed with Inductively Coupled Plasma Emission Spectrometry (ICP-ES). The absorption wavelength and instrument detection limit were 283.553 nm and 0.007 μg/L. For the analyses, the gill, a part of skin and approximately 5 g of each fish stored at -20°C prior to analysis. The digestion of the fish tissues was in accordance with methods described by [13] with concentrated HNO 3 /HCl (1:3 V/V). Metal concentrations were determined with ICP-ES. The concentrations were expressed as micrograms per gram weight (μg/ g. wt.) of tissue in organisms.

Statistical evaluation
For the survival tests, Statistical Analysis of data was carried out with SPSS statistical package program. A value of p < 0.05 was considered to be significant for the accumulation tests.

Results
The concentrations of Cr measured in the water aquariums throughout the experiment were ± 10% of the nominal concentrations (Table 1). Therefore, nominal concentrations from now on will take this value as the basis. The general agreement of Cr 6+ with the Cr remained in the Cr 6+ form throughout the experiment [14]. Tables 1 and 2 summarize the measured Chromium concentration in water (Cr 6+ ug/L) and the survival of the fish during a 4-week-exposure period for juvenile specimens. Survival of control fish was nearly 100% throughout the experiment. Chromium concentrations (25 and 30 mg/L Cr 6+ ) reduced fish survival beginning from the first week of experiment, where high mortality rate of Cr-exposed fish occurred within 14 to 21 days. Survival rate decreased to 52.7c % in fish exposed to 30 mg/L Cr 6+ dose from days 21 to 28 ( Table 2). The behavioral changes of the control group and Tilapia exposed to various doses of K 2 CrO 4 were compared with each other during the experiments. The control group displayed normal behavior during the test period. The lowest concentrations (10 and 15 mg/L Cr 6+ ) had similar behavior to that of the control group. From the dose 20 mg/L Cr 6+, the fish started to show behavioral disorders such as loss of equilibrium, sudden startling and respiratory difficulties. 25 mg/L Cr 6+ and at the highest concentration (30 mg/L Cr 6+ ) onwards, there was shivering, rather high respiratory disorder and swimming in capsized position. The concentrations (μg/ g. wt.) of Cr in the organs fish increased when they were exposed to Cr (Figures 1a-1e). Chromium 1.25 to 12.25 μg/g. wt., while concentrations in the gill were within the range of 3.06 -44.83 μg/ g. wt. (Figures 1a-1e). As can be seen in Figure 1a, maximum level of Cr 6+ was 12.25 μg /g.wt. on fish muscle having lived 28 days at 10 ug/L Cr 6+ initial concentrations. On the other hand, higher concentration of Cr 6+ (12.25 μg/g. wt.) was observed on fish muscle after 28 days at 30 ug/L Cr 6+ (Figure 1e). Maximum levels of Cr 6+ concentrations on the muscle tissues at 15 (after 28 days) in Figure 1b, 20 (after 28 days) in Figure 1c and 25 (after 28 days) ug/L Cr 6+ treatments in Figure 1d were 2.43, 9.03 and 10.81 μg /g. wt., respectively. The concentrations of Cr in gills were almost twice, three and four times as high as in the muscle at 20, 25 and 30 ug/L Cr 6+ mediums, respectively. The Cr accumulation on the skin of Tilapia for all experimental concentrations was nearly twice as much as in the muscle of samples at the end of experiment.

Discussion
It is widely known that metal toxicity is more accurately measured in fresh water than in sea water, because metal appear to a great extent as complex compounds in sea water and this reduces the toxicity of metal ions. In the present study, the mortality increased with an increase in Cr 6+ concentration and also the duration of the exposure to Cr 6+ . This may be rather significant, because smaller fish being generally more active than larger ones, metal uptake and elimination of metal could also occur in higher rates in these smaller ones [15,16]. A comparison of toxicity values for 10 μg/L Cr 6+ to 30 μg/L Cr 6+ experiments demonstrated a decline in survival rate following longer exposure periods. Even though the magnitude of this decline varied between the different chromium concentrations, lower (10 to 20 μg/L Cr 6+ ) mediums were about 10 to 20% mortality while higher (25 and 30 μg/L Cr 6+ ) mediums were 38 to 45% mortality at the end of experiments. Nearly no mortality was recorded in the experiment controls. The Cr concentrations [10 to 30 μg/L (ppb) Cr (VI)] used in the present study has been proved not to be sub lethal for juvenile Tilapia and have been considered as representatives of environmental exposure, fish showed externally abdominal distention and hemorhagic (reddening) and- Different superscript letters (a, b and c) designates difference at p < 0.05 within a sample day.  The damage was noted increasing at the exposure of 30 mg/L onwards. In the lowest concentration through the effect was basement membrane and sub mucosa. The gill filaments and its constituents were damaged gradually which reached to the total collapsed level at the highest concentration (40 mg/L). At the higher concentration, the gill rakers start hypertrophy and that was follow by the hypertrophy in gill filaments by their hypertrophy. All these changes caused the fish to avoid taking food and intake of oxygen. These patterns of changes have also been reported by many workers [24,25]. It was also observed that the heavy metals in muscle tissue were at low levels compared with other organs [26]. The results also show that chromium is more accumulated in the samples of gill than in the skin or muscles [23]. uregenital pours. Erosion and fusion were also detected in lamellar epithelium of gills. Several studies showed the effects on aquatic living things have been evaluated; some data exist on salmon [14].
Other researcher studied heavy metal accumulation in tissues of Tilapia, a freshwater fish [17][18][19][20]. It was noted that Cr accumulation in the tissues showed the following sequence: gills > skin > muscle tissues (least). Other study found that the lowest concentration of Cr was detected in the muscle tissue and skin and the gills showed similar levels (p > 0.05) [21]; chromium in the spleen, muscle and gills; copper in the kidney; zinc in the gills [9]. Another study indicated that concentrations of heavy metals on wild fish were higher in all of the skin samples than in the muscles [22]. The reason for high metal skin complexion with the mucus that is impossible to remove completely from the tissue before analysis. In general, higher metal concentrations in gills reflect metal concentrations of the water where the fish live. Some studies showed that the gills which are very soft part of the body involved in the respiratory process start damaging expose at lowest concentration of Cr (25 mg/L) [23].  Cr Conc Exposure days