Research Journal of Chemical Sciences ______ ______________________________ ______ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 41 - 45 , March (201 2 ) Res.J.Chem.Sci. International Science Congress Association 41 Acute Toxicity of Mercury (HgCl 2 ) to African Catfish, Clarias gariepinus Guedenon P. 1 * , Edorh A.P. 1,2 , Hounkpatin A.S.Y. 1 , Alimba C.G. 3 , Ogunkanmi A. 3 , Nwokejiegbe E.G 3 and Boko M. 1 1 Interfaculty Centre of Training and Res earch in Env ironment for Sustainable Development (CIFRED), University of Abomey - Calavi (UAC), 03 BP 1463, Jéricho, Cotonou BENIN 2 Biochemistry and Cellular Biology, University of Abomey - Calavi (UAC), 01BP 526 Cotonou, BENIN 3 Department of Cell Biology and Genetics, Faculty of Science, university of Lagos, NIGERIA Available online at: www.isca.in (Received 1 5 th January 2012 , revised 30 th January 2012 , accepted 7 th Februry 2012 ) Abstract In order to assess the acute toxicity of mercury on Clarias gariepinus, 108 fish of mean weight 51.27 g ± 2.01 and mean lengt h 20. 2 cm ± 0.72 were divided into six groups of six fish each. The different groups were exposed to the different conc entrations of 0 mg/L, 0.3 mg/L, 0.5 mg/L, 0.8 mg/L, 1 mg/L et 1.50 mg/L for a period of 96 hours. The experiment was triplicated. The results revealed that all the fish of groups exposed to 0 mg/L of HgCl 2 (control) survived whereas all the fish of groups exposed to 1 mg/L and 1.5 mg/L died. The determination of 96 hours LC50 was carried out by computing the mortality results in Probit program of SPSS (version 17.0). The median lethal concentration was 0.60 mg/L with lower and upper confidence limits of 0.1 35 mg/L and 3.519 mg/L respectively at 95%. Keywords : Clarias gariepinus, mercury, acute toxicity, lethal concentration . Introduction In modern times, one of the main threats to the health of ecosystems is the exposure to a myriad of toxic substances and compounds such as mercury, cadmium, lead, copp er, arsenic, air pollutants, pesticides, plastics, cigarette smoke, diesel fumes and nano - particles found in products like perfumes and sunscreens. Because of their high toxicity conferred by their persistent nature in the environment, heavy metals come to the forefront of dangerous substances causing serious health hazard in ecosystems and organisms 1 - 3 . Their introduction into aquatic environment is caused by direct or indirect agricultural and industrial discharges. Heavy metals contamination could b e detrimental to ecological balance of the recipient environment and to a diversity of aquatic organisms 4,5 . Since fish are animals particularly affected by these pollutants they are widely used to evaluate the health of aquatic ecosystems 6 . Some particular heavy metals, such as mercury (Hg), are especially of a deep concern due to their high toxicity. Mercury occurs naturally as a mineral and is widely distributed throughout the environment as a result of natural and human activities. Inorgan ic mercury is the most common form of the metal released by industries in the environment 7 . Once in the aquatic ecosystem, part of the inorganic mercury can be microbiologically converted into methyl - mercury. The resulting organomercury compounds are rapi dly absorbed by the gastrointestinal tract where 90% of ingested mercury is directly absorbed by fish from water through gills, skin and digestive tract 8 . Once contaminated by Hg, fish suffer pathological alterations, with consequent inhibition of metaboli c processes, haematological changes, and decline in fertility and survival 9 . The aim of this study was to determine the acute toxicity of mercury chloride to Clarias gariepinus. C gariepinus was chosen as an experimental animal on the basis of important criteria. The organism is a representative species and widely used in aquaculture throughout Africa. Moreover it is largely tolerant of high concentration of heavy metals. In addition, Clarias gariepinus is a hardy fish and can survive difficult conditions. Material and Methods One hundred and fifty juveniles of Clarias gariepinus of the same brood were purchased from a reputable fish farm (Yanaplaza) in Lagos and transported in an oxygen bag to the aquaculture laboratory of the Faculty of Science of University of Lagos. Their mean weight and length were 32 g ±1.82 and 18.2cm ± 0.61 respectively. The fish were kept in plastic tanks (30x 30x60cm) which were half filled with dechlorinated water. They were acclimatized to laboratory conditions over six weeks. During acclimatization, the juveniles were fed with commercial fish feed known as Coppens at 4% of their body weight. Proximate composition of Coppens is shown in table 1.The Juveniles were f ed thrice daily (morning, afternoon and evening) and the water was changed every twenty four (24) hours to prevent the accumulation of waste metabolite and food particles. They were kept at 12 hours of photoperiod. During the whole period of acclimatizatio n, the mortality recorded was below 2%. At the end of acclimatization the new mean weight and length were 51.27g ± 2.01 and 20.2 cm ± 0.72 respectively. The juveniles were randomly divided into six groups of Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 41 - 45 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 42 eighteen fish and each group subdivided into a s et of three subgroups consisting of six fish each. The eighteen subgroups were kept in eighteen different tanks in 15 liters of water. The six groups were respectively exposed to 0 mg/L, 0.3 mg/L, 0.5 mg/L, 0.8 mg/L, 1.5mg/L and 3 mg/L of mercury chloride (HgCl 2 ). In other words, there were five experimental groups and one control group, each group having three replicates. In respect of the preparation of the stock and test solutions of mercury the test chemical used for the experiment was anhydrous mercu ry chloride. Mercuric Chloride powder (HgCl 2 =271.50; minimum assay Hg: 98%) was purchased from “General Purpose reagent BDH Chemicals Ltd Poolo England”. The chloride form of the metal was chosen because of its lower toxicity compared to the other forms o f mercury 1 0 . After a range finding test, the concentrations prepared for the experiment were 0 mg/L, 0.3 mg/L, 0.5 mg/L, 0.8 mg/L, 1.5mg/L and 3 mg/L of mercury chloride (HgCl 2 ) respectively. A stock solution of 100mg/L (0.1g/l) of the mercury was prepare d by adding 100 mg of mercury to 1 liter of distilled water. The different volumes of the solution stock used are shown in table 2. Twenty four hours (24) prior to the exposure to mercury chloride and during the whole period of exposure of 96 hours the fe eding of the fish was stopped. During our investigation, methods for acute toxicity test recommended by UNEP were implemented 11 . A static renewal bioassay technique in which the test media were renewed at the same concentration once every twenty four (24) hours was adopted 12 . The bowls were covered with mosquito mesh – sized nets to prevent fish from jumping out or move from one bowl into another. The set up was monitored hourly to observe changes in fish behavior and remove dead fish. A fish was considered dead when observed to be totally immobile with no opercula movement seen when probed with a glass rod. With respect to the water used in the experiment, its physico - chemical parameters were measured after exposure of the tap water to air in order to lose chlorine. These parameters included temperature, dissolved oxygen, and the potential of hydrogen (pH). They were measured using Horiba multi - parameter by submersing the sensor in the water at a depth of 10 cm from the water surface. All parameters displac ed on the screen of the apparatus. Table - 1 Proximate centesimal Composition of Coppens 13 Moisture Ash Crude protein Crude lipid Crude fiber 8.2% 9.5% 45% 12% 1.5% With regard to statistical analysis, all the mortality results were treated with the computer statistical package (SPSS, version 17.0) SPSS. Linear regression analysis of Probit program was carried out to determine the median lethal concentration (LC50). A lso the student test was carried out to assess if the differences between experimental groups and control were significant. The difference is regarded as highly significant if P value is lower than 0.01, statistically significant if P value is lower than 0 .05, and non significant if P value is higher than 0.05. Results and discussion Bioassay is a necessity to determine the concentration of a toxicant, which could be allowed in waters without adverse effects on the living organisms 14,15. The physico - chemical parameters of the water measured for were temperature, dissolved oxygen and pH. The results are given in table 3. Table – 2 Preparation of the Toxicant Concentrations Used Required concentration of HgCl 2 (mg/L) Volume of stock solution (mL) used to meet the required concentration Total volume of stock solution (mL) to meet the required solution in 15L A: 0.0 0.0 0.0 B: 0.3 3 45 C: 0.5 5 75 D: 0.8 8 120 E: 1.5 15 225 F: 3 30 450 Notice: Prior to addition of the calculated volume of the toxicant, the same amount of tap water was removed from the tank. Table - 3 The physico - chemical characteristics of the water used Parameters Values Temperature 27 şC Dissolved oxygen 7.44 mg/L 6.5 pH 7.02 6.5 – 8. Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 41 - 45 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 43 The physico - chemical parameters recorded were within the permissive limits fixed by WHO which are 6.5 and 6.5 - 8.0 respectively for dissolved oxygen and pH. The results of mortality of Clarias gariepinus after 96 hours of exposure to mercury chloride are shown in figure 1. It was observed that the mortality recorded in this investigation increased with the rise in concentration. The first death was noticed thirty minutes after the introduction of toxicant in the bowl with the highest concentration in mercury chloride (3mg/L). Olaifa et al. 6 reported the first death three hours after introduction of toxicant in the exposure of Clarias gariepinus to lethal and sub - lethal concentrations of copper. Datta and Kaviraj 16 , Fafioye et al. 17 and Okomoda et al. 13 recorded the first death 36 hours after the exposure to acute toxicity treatments of Clarias gariepinus with Synthetic Pyrethroid Deltamethrin, Parkia biglobosa and Raphia vinifera extracts and F ormalin respectively. Guedenon et al. 18 remarked the first death after thirty hours while treating Clarias gariepinus with 120 mg/L of cadmium sulphate. The duration of resistance of Clarias gariepinus in the present study appeared to be the lowest compared to those in the aforementioned studies. Although Clarias gariepinus has proved to be very resistant to various toxicants 6, 13, 16 - 18 , it has shown very little resistance to mercury. In the tanks wit h 1.5 mg/L and 3 mg/L concentrations of mercury all the fish died. However no death was recorded in the control tank devoid of mercury. This observation confirmed that the mortality registered could entirely be attributable to the chronic effects of mercur y. The estimation of the lethal concentration values (LC 50) was carried out using the linear regression of Probit program. The result is shown in figure 2. Figure - 1 Graph depicting the evolution of mortality with the varying conc entrations Figure - 2 Lethal concentration (LC50) of Clarias gariepinus exposed to mercury 0 2 4 6 8 10 12 14 16 18 20 0 0.5 1 1.5 2 2.5 3 3.5 Mortality of Clarias gariepinus Exposure concentrations of mercury chloride (mg/L) Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 41 - 45 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 44 From the graph of figure 2 the 96 - hour LC50 value was determined to be 0.60 mg/L with lower and upper confidence limits of 0.135 mg/L and 3.519 mg/L respectively at 95%. The LC50 found in this investigation is similar to that of Hirt and Domitrovic 19 in th e exposure of Aequidens portalegrensis to acute concentrations of mercury chloride. Slabbert and Venter 20 reported LC50 of 0.200 mg/L in the treatment of Poecilia reticulate with mercury chloride. Shyong and Chen 21 found LC50 values of 0.168 mg/L and 0.1 61 mg/L in the exposure of Variocorhinus barbatulus and Zacco barbata respectively. Ishikawa et al . 22 recorded 0.22mg/L in an acute mercury toxicity treatment to Oreochromis niloticus . The median lethal concentration in our study was the highest recorded compared to those reported in the aforementioned investigations. The chemical product being the same, the difference in the results could be attributable to the variety in species used . Here, Clarias gariepinus proved to be more resistant to mercury chloride than the various species involved in the studies already mentioned. However the LC50 found in the present study was by far lower than those reported with Clarias gariepinus by Ayub a and Ofojekwu 23 , Ezike et Ufodike 24 , Lawson et al . 25 and Guedenon et al . 18 which are respectively (204.17 mg/L) for Datura innoxia , (3 , 34mg/L) for petrol, (1,29mg/L) for Lindane (Gamma Hexachloro - Cyclohexane) and ( 46,11mg/L) for cadmium sulphate. The difference might be due not only to the various substances and compounds used in the experiments but also the distinct environmental conditions. Behavioral changes were observed in the catfish in the poisonous solutions. Those symptoms were hyper - activity and attempts to jump out due to skin irritation, restlessness, respiratory distress, loss of balance, gulping for air due to respiratory rate impairment, darkening of the body, sudden and quick movement, rolling movement, back stroke, excessive accumulati on of mucus, all these ending in death. The reaction to the toxicant was more noticeable in the media containing the highest two concentrations of mercury chloride. These observations accord with those remarked by Hirt and Domitrovic 19 , Oti 26 , Oshode et a l, 27 , Ezike et Ufodike 24 and Guedenon et al . 18 during acute toxicity tests. The accumulation of mucus on the body could be connected to the intensification of mucus secretion of mucous cells activated by the toxicants 28,29 . The ensuing death might be due to increased heart failure, hypertension, gastric hemorrhage, convulsion, paralysis, heart failure and suffocation 30 . Conclusion The investigation of exposing Clarias gariepinus to mercury highlighted the high toxicity of mercury by the mortality recorde d in the poisonous fish and hence a particular attention should be given to the use of products containing mercury. 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