INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 6, Copyright by the authors - Licensee IPA- Under Creative Commons license 3.

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 6, 2013 Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article ISSN Skin permeability induced absorption of metals under fluoridation in edible fish Notopterus notopterus, Farha Aziz 1, Rafia Azmat 2, Farah Jabeen 1 1-Department of Biochemistry, Jinnah University for Women, V C Nazimabad, Karachi 74600, Pakistan 2-Department of Chemistry, University of Karachi, Pakistan farahtariq2@hotmail.com doi: /ijes ABSTRACT The skin of fresh water edible fish Notopterus notopterus was analyzed for the determination of essential and nonessential metals under fluoride stress. Levels of selected metals like Lead (Pb), Mercury (Hg), Magnesium (Mg), Iron (Fe), Zinc (Zn), Sodium (Na), Calcium (Ca), Potassium (K), Fluoride (F), and Chloride (Cl) at different time intervals with fluoride at 1.5 g and 3 g in the skin of Notopterus notopterus from Keenjhar lake, Sindh, Pakistan, were determined using Perkin Elmer Atomic Absorption Spectrophotometer. It was found that all metal concentration was increased as compare to control group. The trend of accumulation of selected metals in the skin at 1.5 g of fluoride for 24 h Na > Mg; At 48 h Na, Ca, Mg > Pb, Fe > K > Cl; At 72 h Mg > F > Ca > Cl > Pb > K. The trend of accumulation of selected metals in the skin at 3 g of fluoride at 24 h Pb >Mg; At 48 h Na > Mg > Fe > Cl, Ca > Pb > & Hg, F; At 72 h Mg > Cl > Ca > Pb, Na > Hg & K. Keywords: Essential and Non-essential metals, Fluoride, Skin. 1. Introduction Fluoride is well known strong, hard anion and accumulative toxic mediator (Guney et al., 2007) occurs naturally and widely distributed in the rivers, lake and seas of world (Azmat, 2009). Fluoride has an affinity to bio accumulate permanently, in the long bones of vertebrates, causing fluorosis, when present in excessive amounts (O'Riordan, 1990). Fluoride is a cumulative toxic agent like lead, arsenic and cadmium. It is most active damaging environmental pollutant and may be considered as genotoxic, neurotoxic and mutagenic agent and can be able to induce genotoxic, neurotoxic and mutagenic effects on fish. (Mclvor, 1990; Bhatnagar et al., 2005; Azmat et al., 2007; Tripathi et al., 2009). Research studies showed that inorganic fluoride ions are directly toxic to aquatic and terrestrial life and easily accumulate in the tissues especially fish, at concentrations where absorption rates are greater than excretion rates because of their environmental persistence and their tendency for bioaccumulation (Neuhold and William, 1962; Foulkes and Anderson 1994; Gosh and Adhikari, 2006; Masoud et al., 2006). Fluoride acts as enzymatic inhibitor disturbing enzyme activity and, finally, interfering metabolic processes such as glycolysis and synthesis of proteins (Camargo, 2003). It may be xenobiotic to the biological system at elevated level. Xenobiotic are generally very lipophilic and need to undergo biotransformation to be converted into more water-soluble compounds that allow them to be excreted from the body (Shi et al., 2009). Fluoride is the most electronegative and active nonmetallic element of the periodic table, with very high electro negativity and reactivity (Suwalsky et al., 2004). The intake of fluoride by ingestion, inhalation and absorption Received on May 2013 Published on August

2 through the skin, damage the cellular structure which results in accumulation of other toxic metals in the body which is real danger to the life because metals showed adverse effects on bio molecular structures of tissues (Handy and Eddy, 1990). The rate of absorption of oxygen through gills and skin depends on the availability or fluoride concentration. Higher the fluoride concentration lowers the absorption of oxygen which will be later on produced alteration in all biochemical processes occurring in fish. Shi et al (2009a, b) reported significant increase in fluoride concentration in bone, cartilage, skin and gill of Siberian sturgeon exposed to lethal dose of fluoride. The skin together with the gills of aquatic vertebrates act as the outer most defense barrier against the surrounding environmental pathogens and toxicants (Ojha, 1997; Esteban, 2012). The mucosal surfaces of fishes (gill, skin, and gastrointestinal tract) form a thin physical barrier between the external and the internal environment and are considered the first line of defense against contamination through skin epidermis (Shephard, 1994; Woof and Mestecky, 2005). Mineral nutrients are vital for growth, development, and reproduction of all living organisms. Minerals misbalancing of Sodium (Na), Magnesium (Mg), Iron (Fe), Zinc (Zn) and others by toxicants may disrupts numerous physiological system in living system (Kirkby, et al., 1996; Adame, 2002; Skinner, 2005).Micronutrients interact with toxic substances at several sites in the body, increase adaptation and excretion of electrolytes, transport non- essential substances across the membrane and binding to target proteins. As a metabolic toxin it stimulates some enzymes of metabolism, sequestration of toxic metals and finally accelerates the secondary mechanism of toxicity such as oxidative stress (Mclvor, 1987; Valavanidis and Vlachogianni, 2010). Fluoride is highly mobile inorganic pollutant and presence of fluoride may increase the permeability of cell membrane in such a way it provides help in accumulation of essential and non-essential metals in the organ that directly exposed to toxicants such as skin and gills. In addition any increase and decrease of the bioavailability of micronutrients metals adversely affect the physiological functions of various organs (Azmat et al., 2011). This research study has been designed to study accumulation of essential macronutrients and micronutrients in skin of edible fresh water fish Notopterus notopterus in presence of fluoride in relation with the permeability of skin tissue. 2. Material and methods All chemicals and reagents used were of analytical grade (Merck, BDH, UK) Fish sampling site Healthy living fish N. notopterus were collected from the fish farming region of Keenjhar Lake, Thatta, Sindh, during March 2010 Figure -1. Temperature of lake was 30 O C. Humidity 70%. Fish were caught with the help of professional local fisherman by using local fishing nets and motor boat Figure Fish assimilation in laboratory Fish were transported to laboratory under ordinary conditions. Fish were grouped and placed in a fiber glass aquarium size: 36cm x 18cm x 15cm containing tap water. Air pumps and filters were used to aerating the aquarium water. All control and treated fish groups were feed with commercial pellet once a day. Water in aquaria was changed after two days. The fish were divided into three groups with ten fishes per group. Group 1 serves as non-treated while group II and III served as experimental groups. Group II were treated with sub- 2340

3 lethal concentration of fluoride 1.5gm / 70 L of NaF and group III were treated with lethal concentration of fluoride 3gm/ 70 L of NaF. Both control and treated fish were scarified after 24, 48 and 72 h. Both non-treated and treated fish were dissected by using dissection kit. Skin of non-treated and treated were removed and washed with distilled water and blotted with blotting paper. All skins were placed in sterile labeled polythene begs and incubated in freezer at -20 C for further chemical analysis. Figure 1: Fish farming zone in Keenjhar Lake Figure 2: Fish were caught with the help of professional local fisherman 2.3. Preparation of Skin Sample for Macro & Micro Nutrients Analysis by Digestion Method Equal weights of all skins including control and treated fish were put into crucibles (using 0.2 g dry mass and a dilution factor of 50). The crucibles were placed in the oven for 2 h at 135 ºC. After that the samples were mineralized at 400 ºC in the chemical oven for 24 h then 2 ml of nitric acid was added and sampled were dehydrated at 450 ºC. To each sample 10 ml of hypochlorous acid was added and then make up to 50 ml with double distilled water. The detection of macronutrients, micronutrients and heavy metals were carried out by atomic absorption spectrophotometry with graphite furnace and acetylene flame. 2341

4 2.4. Statistical analysis The data was subjected to statistical analysis. Student s t- test was used to determine the level of significance of the heavy metal concentration in control and treated fish. Differences were regarded as significant at p Results and discussion Fluoride is a widespread, non-biodegradable, highly oxidizing element of the periodic table with four electronegativity and relatively persistent contaminant of the environment (Lee J, 1983). It is also classified as persistent inorganic pollutants that can bio accumulate in the environment and food chain. The analysis of essential and non-essential metals was carried out after regular time intervals of 24, 48 and 72 h at two different sub lethal concentration of sodium fluoride (1.5 g/70l and 3 g/70l) in fresh water fish N. notopeterus collected from Keenjhar Lake, Thatta, Sindh. This research work specifically focus on essential and nonessential metals accumulation trend under fluoride stress in skin of fish N. notopeterus along with bio-concentration and bio-magnification methods in relation with skin permeability. Fluoride is well-known as a most important biologically active and mobile toxicant. Mineral metal elements play an important role in the growth, development, reproduction and maintenance of various tissues. Mineral such as calcium is needed for the contraction of muscles and the functioning of the nerves. Skin tissue were examined after 24, 48 and 72 h for essential macro-nutrients( Na, K, Ca and Mg) and micro-nutrients (Hg, Pb, Cl and Fe) as first site of interaction of any toxicants of aquatic resources. The results are summarized in the Tables 1-2 and Figure 2-3. It was observed that skin showed much more variation in macro-nutrients at 24h, 48h and 72h at both doses (1.5g/70L & 3g/70L) of fluoride. Micronutrients showed an elevated results at all exposure times and dose. No mortality was observed during whole trial period Effect of Fluoride on Macro elements (Na, K, Ca and Mg) of Skin The Sodium and Potassium content at sub lethal concentration of fluoride (1.5g/70L) was found to be increased at all exposure time as reported in the Table 1. The sodium content at high sub lethal concentration of fluoride (3.0 g/70l) was found to be decreased within 24 h and 72h of exposure. The decrease in concentration of Na depends upon the time of exposure and the concentration of fluoride as reported in the Table 2. Table 1:Effect of fluoride (1.5g/70L) on Macro elements (ppm) in skin of fresh water N. notopterus at 24, 48 and 72 h; temp. 30ºC, ph= 7.8 Macro elements Control 24h 48h 72h Na 30.2± ±0.233* 41.1±0.233*** 42.9±1.015** K 17.6± ±0.221*** 22.1±0.233*** 31.9±0.233*** Ca 22.2± ± ±0.260** 37.7±0.213*** Mg 8.4± ±0.249** 19.8±0.249*** 29.6±0.213*** Values expressed as Mean ± S.E.M; p = probability, p = probability, *** represent highly significant (p<0.001), ** represent significant (p<0.01), * represent significant (p<0.05) compared with control, ND = not detected 2342

5 Table 2: Effect of fluoride (3g/70L) on Macro elements (ppm) in skin of fresh water N. notopterus at 24, 48 and 72 h; temp. 30ºC, ph= 7.8 Macro elements Control 24h 48h 72h Na 30.1± ±0.223** 41.2±0.200*** 27.8±0.249** K 17.7± ± ±0.305*** 17.7±0.300 Ca 22.0± ±0.260** 37.7±0.213*** 45.1±0.233*** Mg 8.4± ±0.223*** 16.6 ±0.221*** 40.6±0.221*** Values expressed as Mean ± S.E.M; p = probability, p = probability, *** represent highly significant (p<0.001), ** represent significant (p<0.01), * represent significant (p<0.05) compared with control, ND = not detected The Potassium content in skin was not adversely affected by the applied fluoride dose when compared with the control. Sodium and Potassium are the major positive ions in body fluid that regulates the total amount of water in the body in term of osmotic pressure. Important physiological processes in the body, especially in the brain, nervous system, and muscles, require sodium and potassium for the generation of electrical signals. Minerals misbalancing can cause malfunctioning of metabolic tissues and may be fatal to whole system (Azmat et al., 2011). Decrease and increase in concentration of these essential nutrients disturb the normal functioning of skin tissues and these effects were observed visually as a discoloration of skin of treated fish as compared to control one.the Calcium (Ca) and Magnesium (Mg) contents under fluoridation at 1.5g/70L & 3.0 g/70l was found to be increased at all exposure time as reported in the Table-1-2. Which may be due to the adaptation for protection of skin tissues as calcium is stored in the sarcoplasmic reticulum of muscle cells as the bioavailability of fluoride is reduced by increasing level of calcium content (Gosh, 2006; Foulkes and Anderson, 1994) Increase in calcium contents may affect fish behavior or tends to increase the resistance of fish to fluoride or it may be due to the reaction between fluoride and calcium. Magnesium is known as anti-stress mineral, increases the resistance against stress, depressions and tensions. It also involved in releasing energy from the diet and is involved in a normal functioning of nervous system and muscles. Magnesium helps your heart maintain its normal rhythm (Azmat et al., 2011). Increase in magnesium content may be associated with resistance produced by fish under applied fluoride stress or to decrease the permeability of skin exposed to fluoride. A positive correlation (p < 0.001) in between macro elements and fluoride may showed the macro-minerals a significant misbalancing which results in the distortion of skin tissues. Fluoride due to its high biological activity and small ionic radius can penetrate easily into the organisms and tissues. It has adverse chronic effects on bio molecular structures of different tissues (Rosenquist, Lemperg, 1974; Devi, Piska, 2006; Kaushal, Mishra, 2011). Due to its higher electronegativity it frequently oxidizing the C-C linkages of protein, lipid and other important macro and micro nutrient, due to which variations in skin color and fish behavior were observed Effect of fluoride on micro elements of skin Effect of fluoride at two doses (1.5g/70L & 3g/70L) was studied on micro nutrients (Pb, Hg, Fe, Cl) in skin of fresh water fish N.notopterus collected from Keejhar Lake, Thatta, Sindh. 2343

6 Results were reported in the Figure 1-2 and showed that the mineral imbalances reveal the chronic effects of fluoride. Figure 1: Micro element (Pb, Hg, Fe, Cl )) contents at different time intervals under fluoride stress(1.5g NaF/70 L) Figure 2: Microelement (Pb, Hg, Fe, Cl) contents at different time intervals under fluoride stress (3g NaF/70 L) The Chloride content at sub lethal concentration of fluoride (1.5g/70L & 3.0 g/70l) was found to be increased at all exposure time whereas Iron content was elevated at both doses at all exposure time as reported in the Figure-1-2. Chloride ions (Cl-) are used to regulate appropriate ph levels in blood and maintain the body's acid-base balance (homeostasis). Chloride moves primarily with sodium and water and helps create the osmotic pressure of body fluids. Result showed Iron (Fe) content was elevated at sub-lethal concentration of fluoride (1.5g/70L & 3g/70L). Iron is required for the production of red blood cells, process known as hematopoiesis. It is also part of hemoglobin binding to the oxygen and thus facilitating its transport from the lungs via the arteries to all cells throughout the body. Iron is also involved in the conversion of blood sugar to energy. The production of enzymes, which play a vital role in the production of new cells, amino acids, hormones and neurotransmitters, also depends on iron. The immune system is dependent on iron for its efficient functioning and physical and mental growth. 2344

7 Toxic metals like Mercury (Hg) and Lead (Pb) accumulation in the skin of edible fish Notopterus notopterus increases with an increase in the concentration of fluoride. These results suggest that fluoride facilitate the trace metals concentration in skin tissue which are non-degradable and regarded as hazardous to aquatic ecosystem because of their environmental persistence and their tendency for bioaccumulation (Gosh and Adhikari, 2006). 4. Conclusion Fluoride is a cumulative poison and the most damaging environmental pollutant may be considered as mutagenic which can induced or increase the occurrence of mutation of an organism. Fluoride ions are directly toxic to aquatic life and accumulate in the tissues, at concentrations where absorption rates exceed emission rates whereas the concentration of fluoride directly affect the aquatic organism by accumulating many trace metals in vital part of fish. Research on the macro and micronutrients by toxicants caused minerals misbalancing which may result in the alteration of physiological processes of organs and adversely affect the function of the organs. These results clearly demonstrate that permeability in skin tissue due to fluoride exposure result in entering various toxic metals in fish under study, which may cause detritus changes with disrupt tissue structure at molecular level that ultimately decrease the absorption of oxygen in skin results in death of fish at long exposure. Acknowledgments This study was supported by institutional funds of Jinnah University for Women, Karachi- Pakistan.We are thankful to Aliya Begum, Dura - Shahwar and Mohammad Imran for their laboratory and technical assistance. 4. References 1. Adame. L., (2002), Leaf absorption of mineral nutrients in carnivorous plants stimulates root nutrient uptake. New Phytologist, 155, pp Azmat. R., (2009), Natural Chemical Technology for reducing Fluoridation through chloride ions in Fish of Sindh Region. Journal of Chemistry and Chemical Engineering, 3, pp Azmat. R; Talat R; Khalid A., (2007), The length-weight relationship, Condition factor and impact of fluoride concentration in Johnius belangerii of Arabian Sea. Research Journal of Environmental Toxicology, 1, pp Azmat. R, Aziz F and Bibi S.S, (2011), Accumulation of metals in Gill tissues of N.notopeterus under fluoride stress. Asian Journal of Chemistry, 23(5), pp Bhatnagar. C and Regar BC., (2005), Neurodegenerative effect of Fluoride (NaF) on the brains of fresh water teleost, Labeo rohita. Indian Journal of Environmental Sciences, 9, pp Camargo. J.A., (2003) Fluoride toxicity to aquatic organisms: a review. Chemosphere, 50, pp

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