1. Effects of Fluoride Ion on Bone Topography and its Concentration in Osseous Tissue of Chronically Exposed Atlantic Salmon (Salmo salar) Shawn McGinley, Department of Biology, York College of Pennsylvania Introduction Fluoride is found widely throughout nature and is crucial for the growth, production and mineralization of tooth and bone tissue (Julshamn et al., 2004). Because of its high affinity for calcium, fluoride are typically localized in hard tissues. While vital in small amounts, large doses of fluoride is considered to be toxic (Kotecha et al., 2012) and may result in a chronic systematic disease known as fluorosis (Sun et al., 2013). A universal standard for fluoride levels in drinking water has not been set, however the US EPA has set the maximum level at 4.0 ppm and WHO has set it at 1.5 ppm. Fluoride levels are typically around 1.3 ppm in seawater (Rix et al., 1976) and various levels in ground waters depending on the type of bedrock or level of pollution. Many countries have naturally high levels of fluoride in their ground and surface waters, leading to increased cases of fluorosis. Most fish farms utilize local water sources to house their fish (Shi et al., 2009) which could have negative effects on the fish if high levels of fluoride are present. Several studies report the negative side effects that fluoride has on fish, but the majority utilize food as the exposure medium. Most studies have studied the effects of dietary fluoride on several species of fish, but very few have utilized water as an exposure media. An increasing number of studies have found correlations between fluoride exposure and negative side effects in fish. Aquatic organisms are anticipated to accrue fluoride concentrations relative to their environment. This experiment will help to further elicit the effects that fluoride may have on the organisms that inhabit water sources with varying concentrations of fluoride. The study also proposes a new method for detecting the effects of fluorosis utilizing an atomic force microscope. Commercially, this research will help fisheries produce the healthiest fish possible for release into the wild or as a source of food for humans. Review of Literature Julshman et al. studied the effects of dietary fluoride in Atlantic salmon using Antarctic krill as a source of biological fluoride. Standard fish diets were supplemented with 10%, 20% and 30% krill meal. After 12 weeks, analysis of the muscle, vertebra and whole body was conducted. There were no negative side effects found, suggesting inorganic forms of fluoride, such as those found in water, are more readily absorbed (2004). Cao et al. (2013) utilized carp to determine the effects that varying concentrations of fluoride in water had on its distribution in several tissues and toxicity in the gills. The concentration of fluoride in the brain, gills, intestine, kidney, muscle and liver were analyzed and as concentration in the water and exposure time increased, the concentration of fluoride in each tissue increased as well. Fluoride replaces OH ions in bone apatite (Mehta et al., 1995). Sogaard et al. treated sexually mature rats with sodium fluoride through their drinking water. A significant increase in bone ash density and bone volume for the highest exposure group. The change in structure caused by the addition of fluoride and the removal of hydroxyl ions weakens the integrity of the bone (1995) Proposed Methods Expected Results http://www.krisweb.com/krissheepscot/krisdb/html/kr isweb/aqualife/ats_adult_photo_masc.jpg Literature Cited Cao, J., Chen, J., Wang, J. Wu, X., Li, Y., Xie, L. 2013. Tissue distributions of fluoride and its toxicity in the gills of a freshwater teleost, Cyprinus carpio. Aquatic Toxicology 130: 68-76. Julshamn, K., Malde, M.K., Bjorvatn, K., Krogedal, P. 2004. Fluoride retention of Atlantic Salmon (Salmo salar) fed krill meal. Aquaculture Nutrition 10: 9-13. Kotecha, P.V., Patel, S.V., Bhalani, K.D., Shah, D., Shah, V.S., Mehta, K.G. 2012. Prevalence of dental fluorosis & dental caries in association with high levels of drinking water fluoride content in a district of Gujarat, India. Indian Journal of Medical Research 135: 873-877. Milovanovic, P., Djuric, M., Rakocevic, Z. 2012. Age-dependence of power spectral density and fractal dimension of bone mineralized matrix in atomic force microscope topography images: potential correlates of bone tissue age and bone fragility in female femoral neck trabeculae. Journal of Anatomy 221: 427-433. Rix, C.J., Bond, A.M., Smith, J.D. 1978. Direct Determination of Fluoride in Sea Water with a Fluoride Selective Ion Electrode by a Method of Standard Additions. Analytical Chemistry 48: 1236-1239. Sogaard, C.H., Mosekilde, Li., Schwartz, W., Leidig, G., Minne, H.W. Ziegler, R. 1995. Effects of Fluoride on Rat Vertebral Body Biomechanical Competence and Bone Mass. Bone 16: 163-169. Sun, L., Gao, Y., Liu, H., Zhang, Wei., Ding, Y., Li, B., Li, M., Sun, D. 2013. An assessment of the relationship between excess fluoride intake from drinking water and essential hyptertension in adults residing in fluoride endemic areas. Science of the Total Environment 443: 864-869. Acknowledgements I would like to thank Dr. Rehnberg for his assistance and continued support throughout this process. http://palaeos.com/vertebrates/b ones/dermal/opercular.html Hypotheses 1.Accumulation of fluoride in osseous tissue and bone surface roughness will increase with the concentration of fluoride in water. 2.Accumulation of fluoride in osseous tissue and bone surface roughness will be greater in freshwater than in seawater. Figure 1. The skull bones of a bony fish. The top right bone highlighted in red is the opercular. Figure 4. Atomic Force Microscope image of bone surface (Milovanovic et al., 2012) 180 Atlantic Salmon 90 Salmon (land- locked) 90 Salmon (anadromous) Fluoridated fresh water Fluoridated seawater 30 Salmon – 3 tanks of 10 fish Control Standard (4.0 ppm ) High (25.0 ppm) Anesthetize w/ MS222 Remove Bone Ash bone overnight @ 550 o C Test for fluoride levels using Fluoride ion selective electrode Prepare opercular bone sample for AFM Analyze bone topography with AFM Control Standard (4.0 ppm) High (25.0 ppm) 30 Salmon – 3 tanks of 10 fish 90 day exposure period