Associations Between Omega-3 fatty Acids, Selenium Content, and Mercury Levels in Wild-harvested Fish from the Dehcho Region, Northwest Territories, Canada Ellen S. Reyes 1 , Juan J. Aristizabal Henao 2 , Katherine M. Kornobis 3 , Rhona M. Hanning 1 , Shannon E. Majowicz 1 , Karsten Liber 4 , Ken D. Stark 2 , George Low 5 , Heidi K. Swanson 3 , Brian D. Laird 1 1 School of Public Health and Health Systems, University of Waterloo, Waterloo, ON, Canada; 2 Department of Kinesiology, University of Waterloo, Waterloo, ON, Canada; 3 Department of Biology, University of Waterloo, Waterloo, ON, Canada; 4 Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada; 5 Aboriginal Aquatic Resources and Ocean Management, Hay River, NWT, Canada Fish provide a rich variety of important nutrients [e.g. omega-3 fatty acids (n-3 FAs) and selenium (Se)]. The intake of n-3 FAs from fish consumption promotes healthy growth and development in infants and children (SanGiovanni & Chew, 2005), supports optimal cognitive health in older adults (Dangour & Uauy, 2008), and reduces the risk of cardiovascular disease (Calder, 2004). The intake of the essential trace element, Se, is important to maintain thyroid hormone metabolism (Arthur, 1991), potentially protect against cardiovascular disease (Rayman, 2012), and may reduce the incidence of some cancer types (Vinceti et al., 2014). However, methylmercury (MeHg), a contaminant commonly detected in fish, is known to induce adverse health effects in neurological, cardiovascular, immune, and endocrine systems. To promote traditional food use as a pathway to health equity, the following research objectives have been developed: 1. To quantify the levels of total Hg, n-3 FAs, and Se in fish species harvested from three lakes in the Dehcho Region, Northwest Territories (NWT) 2. To evaluate the correlations between nutrient and contaminant concentrations and identify which fish species provide the most n-3 FA and Se relative to their Hg content The authors gratefully acknowledge the support of the Aboriginal Aquatic Resources and Management Program, Northern Contaminants Program (jointly funded by Aboriginal Affairs and Northern Development Canada and Health Canada), Health Canada’s Climate Change Adaptation Fund, Cumulative Impacts Monitoring Programs, and the School of Public Health and Health Systems at the University of Waterloo for completion of this research. LITERATURE CITED 1. Arthur, J.R. 1991. The role of selenium in thyroid hormone metabolism. Can J Physiol Pharmacol. 69, 1648-1652. 2. Calder, P.C. 2004. N-3 fatty acids and cardiovascular disease: Evidence explained and mechanisms explored. Clin Sci (Lond). 107, 1-11. 3. Dangour, A.D., Uauy, R. 2008. N-3 long-chain polyunsaturated fatty acids for optimal function during brain development and ageing. Asia Pac J Clin Nutr. 17 Suppl 1, 185- 188. Data Collection  Seven species of freshwater fish included: Burbot (Lota lota), Cisco (Coregonus artedi), Lake Trout (Salvelinus namaycush), Lake Whitefish (Coregonus clupeaformis), Longnose Sucker (Catostomus catostomus), Northern Pike (Esox Lucius), and Walleye samples (Sander vitreus)  Sample collection time frame: August 2013  The samples were collected from Ekali, Sanguez, and Trout Lakes in the NWT Laboratory Analysis  Mercury Analysis . Fish tissue samples were freeze dried without skin and ground prior to analysis. Analysis for Hg was measured via a direct mercury analyzer (Milestone DMA-80).  Omega - 3 Fatty Acids Analysis . 100 mg of pulverized fish muscle tissue was homogenized with 2 mL CHCl 3 : 1 mL MeOH containing 22:3n-3 ethyl ester as the internal standard. 50 µg/mL of butylated hydroxytoluene was also included in the extraction reagents to prevent oxidation. The sample was centrifuged and the organic extract was evaporated under N 2 gas by 300 µL of hexane. Analysis for the lipid extracts was measured by a gas chromatograph with a flame ionization detector (Varian 3900). Table 1. Total Mercury and Selenium Concentrations by Fish Species Table 2. Fatty Acid Composition by Fish Species INTRODUCTION AND RESEARCH OBJECTIVES RESEARCH METHODOLOGY RESULTS AND DISCUSSION 4. Legrand, M. et al. 2010. Methylmercury blood guidance values for Canada. Can J Public Health. 101, 28-31. 5. Lockhart, W.L. et al. 2005. A history of total mercury in edible muscle of fish from lakes in northern Canada. Sci Total Environ. 351-352, 427-463. 6. Rayman, M.P. 2012. Selenium and human health. Lancet. 379, 1256-1268. 7. SanGiovanni, J. P., Chew, E.Y. 2005. The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina. Prog Retin Eye Res. 24, 87-138. Figure 1. Relationship between total mercury concentration and omega-3 fatty acid content in Lake Trout Figure 2. Comparison of nutrient: mercury ratios to the de minimus ratios from Health Canada’s mercury regulatory guidelines [EPA + DHA] [Hg] More nutrients; Less mercury More mercury; Less nutrients  Strong negative correlations were observed between HgT and n-3 FA content for Lake Trout (ρ =- 0.937, P <0.001), Northern Pike (ρ = -0.619, P <0.001), and Walleye (ρ = -0.481, P <0.01)  There were also significant negative correlations between Hg and Se observed for Lake Whitefish (ρ = -0.818, P <0.001), Cisco (ρ = -0.685, P <0.05), and Northern Pike (ρ = -0.410, P <0.05)  To the knowledge of the authors, this represents the first time that researchers have demonstrated strong, negative correlations between Hg and nutrient content in particular fish  From a risk-benefit perspective, the presence of negative correlations can contribute to substantial intra-species variation in nutrient:Hg ratios  A method proposed by Tsuchiya et al. (2008) defined a de minimus intake ratio of 17 mg DHA to 1 µg of Hg exposure, such that individuals consuming fish over this de minimus ratio would be able to meet the Dietary Reference Intake of DHA (100 mg/day) while not exceeding the U.S. EPA reference dose (RfD) for MeHg (0.1 μg/kg/day)  To make the de minimus approach more relevant to the Canadian regulatory context, we applied Tsuichya et al. (2008) method using Health Canada’s Hg toxicological reference value (TRV) for pregnant women and women of child-bearing age (Legrand et al. 2010)  Of the seven freshwater fish species collected in this study, Walleye, Longnose Suckers, Lake Trout, Cisco, and Lake Whitefish, on average, exceeded the de minimus ratio for DHA:Hg (8.3:1)  In contrast, all seven of the species were below the de minimus molar ratio for Se:Hg (13.4:1). However, it is not yet known how high the Se:Hg molar ratio has to be to protect against MeHg’s adverse effects Fish Species n Range (ppm) Mean ± S.D. (ppm) n Range (ppm) Mean ± S.D. (ppm) Burbot 9 0.228 - 0.551 0.317 ± 0.101 6 0.117 - 0.155 0.141 ± 0.013 Cisco 29 0.020 - 0.194 0.057 ± 0.045 10 0.116 - 0.239 0.174 ± 0.042 Lake Trout 13 0.207 - 0.643 0.330 ± 0.153 11 0.078 - 0.140 0.140 ± 0.056 Lake Whitefish 29 0.025 - 0.150 0.073 ± 0.038 15 0.086 - 0.307 0.173 ± 0.069 Longnose Sucker 6 0.086 - 0.127 0.100 ± 0.015 5 0.170 - 0.215 0.187 ± 0.020 Northern Pike 48 0.070 - 3.121 0.551 ± 0.598 28 0.090 - 0.229 0.140 ± 0.032 Walleye 53 0.036 - 1.428 0.415 ± 0.305 22 0.089 - 0.284 0.195 ± 0.039 Mercury Selenium Mercury Concentrations  Predatory fish [including Northern Pike (0.551 ppm), Walleye (0.415 ppm), and Lake Trout (0.330 ppm)] had higher Hg concentrations than observed in benthivorous and planktivorous fish species [e.g. as Cisco (0.057 ppm) and Lake Whitefish (0.073 ppm)]  Hg content increased with fork length and/or weight for most of the test fish species (P <0.001)  According to ANOVA, Hg concentrations differed between lakes for most of the studied species. The highest HgT levels (P <0.05) were observed in Sanguez Lake for Cisco, Lake Whitefish, Northern Pike, and Walleye Selenium Concentrations  In contrast to the Hg results, average Se concentrations were quite similar between species, ranging from 0.140 to 0.195 ppm for Northern Pike and Walleye, respectively  Generally, Se concentrations were not correlated with either fork length or weight  As seen with Hg, Se concentrations within fish species differed between lakes. However, the highest Se contents (P <0.05) for Cisco, Lake Whitefish, Northern Pike, and Walleye were observed in Trout Lake Fish Species n Range (mg/100g) Mean ± SD (mg/100g) Range (mg/100g) Mean ± SD (mg/100g) Range Mean ± SD Burbot 9 68.4 – 127 101 ± 18.2 57.4 – 110 86.3 ± 16.3 `0.45 - 0.60 0.49 ± 0.04 Cisco 12 187 – 551 346 ± 115 145 – 344 224 ± 58.9 0.24 - 0.41 0.30 ± 0.06 Lake Trout 11 258 – 4,375 1,689 ± 1,294 172 – 2,332 965 ± 544 0.28 - 0.62 0.41 ± 0.15 Lake Whitefish 24 215 – 2110 458 ± 401 182 – 1,048 299 ± 187 0.22 - 0.53 0.38 ± 0.09 Longnose Sucker 6 208 – 763 499 ± 198 160 – 372 263 ± 68.6 0.45 - 0.69 0.54 ± 0.10 Northern Pike 37 131 – 836 212 ± 123 113 – 707 176 ± 98.6 0.20 - 0.48 0.34 ± 0.06 Walleye 35 117 – 911 230 ± 148 87.5 – 807 198 ± 131 0.21 - 0.47 0.34 ± 0.06 Total Omega-3 Fatty Acids EPA+DHA Omega-6 to Omega-3 Ratios Fatty Acid Composition  There were substantial differences in fatty acid profiles between fish species: o Lake Whitefish, the most commonly consumed fish in the Dehcho Region, had n-3 FA levels (458 mg/100 g) that were 2.1-fold higher than in some predatory fish species, such as Northern Pike (212 mg/100g) and Walleye (230 mg/100g) o The EPA+DHA concentrations in Lake Trout (965 mg/100g) were up to 11.2-fold higher than Burbot (86.3 mg/100g), Northern Pike (176 mg/100g), and Walleye (198 mg/100g) o All of the fish species analyzed had greater levels of health promoting n-3 FA relative to their pro-inflammatory n-6 FA content  Typically, n-3 FA content was not correlated with fork length and weight; the lone exception was Lake Whitefish (P <0.01)  The highest n-3 FA levels (P <0.05) were observed in Ekali Lake for Walleye, Sanguez Lake for Lake Whitefish, and Trout Lake for Northern Pike 8. Tsuchiya, A. et al. 2008. Fish intake guidelines: Incorporating n-3 fatty acid intake and contaminant exposure in the Korean and Japanese communities. Am J Clin Nutr. 87, 1867-1875. 9. Vinceti, M. et al. 2014. Selenium for preventing cancer. Cochrane Database Syst Rev. 3, CD005195. Several lakes in the Dehcho Region contain fish populations with elevated total mercury (HgT) concentrations (Lockhart et al., 2005). For example, in some inland lakes, the mean Hg concentration of predatory fishes like Walleye, Northern Pike, and Lake Trout approached or exceeded the 0.5 ppm Health Canada HgT guideline. Situations among subsistence populations, including Dehcho residents of who have limited alternatives to fish sources, may face the dilemma of weighing the risks and benefits. Focusing solely on the risks of Hg, however, can overlook the nutritional benefits provided by fish consumption.  Selenium Analysis. 100 mg of pulverized fish muscle tissue was digested with 5 mL of HNO 3 and 1.5 mL of H 2 O 2 . Samples were re-diluted with 3.5 or 5 mL of 2% HNO 3 and filtered. Analysis for Se was measured by an inductively coupled plasma mass spectrophotometer (Thermo X Series II).