Assessing Fish Community Health following the Kingston Fly Ash Spill Anna L. George 1, David A. Neely 1, Shea R. Tuberty 2, Robert J. Mottice 1, Donna M. Lisenby 3, Daniel Jackson 2, Yosuke Sakamachi 2 (1) Tennessee Aquarium, Chattanooga, TN; (2) Appalachian State University, Boone, NC; (3) Appalachian Voices, Boone, NC Fly ash has been documented to affect fishes through direct mortality from alkaline shock, chronic oxidative stress, and bioaccumulation of toxic elements such as selenium. Since January 2009, our team has conducted quarterly fish community sampling to begin assessment of the long-term impact of the Kingston spill on the fish community in Watts Bar Reservoir. Sampling has been conducted by boat electrofishing for 25-minute periods at five sites on the Clinch, Emory, and Tennessee rivers. Sampled fish are identified to species and standard measures are recorded (weight and length). A subset is retained for toxicological analysis, while the remainder is released at that site. Over the past year, ERM 2.0 has ranged from 4-6 species and 6-74 individuals. Of the two upstream sites, ERM 3.5 has ranged from 5-13 species and individuals, and CRM 5.5 has ranged from 9-10 species and individuals. Five km downstream of the spill, CRM 3.5 has ranged from species and individuals, while TRM has ranged from species and individuals. Externally, fishes have displayed elevated rates of lesions and anomalies, particularly gill clubbing. In addition, 11.8% of the bluegills caught at Tennessee River Mile in January 2010 (11 km downstream of the spill site) exhibited exopthalmia (popeye). Tissue from these individuals is being screened for selenium loading. As seasonality greatly affects community structure, we plan to continue sampling until at least January Methods Sampling was conducted at a total of eight sites in Watts Bar Reservoir from January 2009-January 2010 (Figure 1). Fishes were collected by boat electrofishing for 25-minute set periods. Most sites were visited at least three times over the sampling period. After collection, fishes were examined for evidence of morphological anomalies, identified to species, and measured (mm TL). A subset were retained for toxicological analysis; the remainder were released at the site. Species richness, evenness, and the Shannon-Wiener index were calculated for each sampling site. Jaccard’s and Morisita similarity indices were calculated for pairwise comparisons of all sites sampled during January During the January 2010 sampling at TRM 567.6, a random subset of 12 bluegills exhibiting exopthalmia (popeye) were retained for comparison with a random subset of 12 bluegills not exhibiting the condition. These fishes were weighed, and measurements of total length, head length, interorbital width, and maximum eye width were taken with dial calipers. Fish were freeze dried, whole fish homogenized, (75% solids, 25% water), digested 0.5 g with 10 ml OmniTrace 70% nitric acid, microwaved for digestion, and analyzed on a Barian ICPAES. Differences in whole body selenium concentration and condition (weight/length) between these two groups were analyzed using a t-test. Correlations between the extent of popeye or condition index and selenium content were examined separately for the two groups. Introduction Previous studies have documented fly ash effects on fish from direct mortality from alkaline shock, chronic oxidative stress, and bioaccumulation of toxic elements such as selenium (Besser et al. 1996, Lemly 2002, Ali 2004) Long-term impacts of selenium toxicity contributed to the extirpation of 19 of 20 species in Belews Lake, NC (Lemly 2002) Our objective is to begin initial studies into the long-term impact of Kingston ash spill on the fish community in Watts Bar Reservoir. Our secondary objective is to determine if selenium may be a factor contributing to the observed morphological anomalies in sampled fishes. Results Species richness and individual abundances were always lowest at ERM 2.0, the site of the spill. Species evenness was typically highest at ERM 2.0, while Shannon-Wiener indices varied (Tables 1 and 2). Of nineteen sampled sites in the past year, seven have contained at least one fish exhibiting an anomaly, with the overall percentage at that site varying from % (Table 2). Under Jaccard’s Index, seven site comparisons from January 2010 were of low similarity, while 3 were moderate. Under the Morisita Index, comparisons of the community at ERM 2.0 with any other site were of low similarity, while all six other community comparisons were of high similarity (Table 3). The incidence of bluegills exhibiting popeye has risen from 1.5% (July 2009, CRM 3.5) to 11.8% (January 2010, TRM 567.6). The proportion of maximum eye width : interorbital width was significantly higher in fish identified as popeye. Whole body selenium concentrations among the popeye fish ranged from mg/kg dry weight, with a mean of 3.87 mg/kg. Concentrations among the control group ranged from 3.36 to 5.23 mg/kg, with a mean of 3.98 mg/kg. There was no significant difference in either selenium concentrations or condition between these two groups. There was also no correlation between extent of popeye and selenium content or condition index and selenium content. Discussion The fish community at the site of the spill is least similar from all other, demonstrating decreased species richness, evenness, and abundance. Selenium cannot be eliminated as a cause of popeye in sampled bluegills at TRM Due to seasonality, fish movement, large population sizes, and reduced sampling effort, this study will need to continue over many years to fully document the impact of fly ash on the fish community in Watts Bar Reservoir. Jan-09Mar-09Jul-09Jan-10 Total Number With AnomalyPercent Total Number With AnomalyPercent Total Number With AnomalyPercent Total Number With AnomalyPercent ERM %7400% %600% ERM 45500% %2400% CRM % % % CRM % %12500% TRM %9900% % ERM % ERM % ERM % Jan-09Mar-09Jul-09Jan-10 RHERHERHERHE ERM ERM CRM CRM TRM ERM ERM ERM ERM 2.0 ERM 4.0 CRM 5.5 CRM 3.5 TRM ERM ERM CRM CRM TRM Table 1. Calculated species richness (R), Shannon-Wiener Index (H), and species evenness (E) at eight total sites in Watts Bar Reservoir over four sampling periods. Table 2. Total number of individuals, number exhibiting an anomaly, and the percentage of anomalies in the population at eight total sites in Watts Bar Reservoir over four sampling periods. Table 3. Jaccard’s Index (above diagonal) and Morisita Index (below diagonal) of similarity for all collections made during January 2010 sampling. Figure 1. Location of sampling sites in Watts Bar Reservoir. Opercular lesion and algal infection (Cladophora sp.) in largemouth bass (300 mm TL), TRM 567.6, March Bluegills exhibiting exopthalmia (popeye), TRM 567.6, January 2010 Lesion on common carp (670 mm TL), CRM 3.5, March 2009 Acknowledgements: Heather DeGaetano, Lee Friedlander, Matt Hamilton, Dave McKinney, Lyndhurst Foundation, TWRA, World Wildlife Fund Southeast Rivers and Streams Program Literature Cited: Ali M, Parvez S, Pandey S, Atif F, Faur M, Rehman H, Raisuddin S Fly ash leachate induces oxidative stress in freshwater fish Channa punctata (Bloch). Environ. Int. 30(7): Besser J, Glesy J, Brown R, Buell J, Dawson, G Selenium bioaccumulation and hazards in a fish community affected by coal fly ash effluent. Ecotox. and Env. Safety 35(1):7-15. Lemly A Symptoms and implications of selenium toxicity in fish: the Belews Lake case example. Aqua. Tox. 57(1-2):39-49.