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1 MEMO E. th Avenue PO Box 7 Helena MT 9-7 () -8 FAX () -9 To: Tracy Stone-Manning, Montana Department of Environmental Quality CC: Eric Urban, Kyle Flynn, & Mark Bostrom Montana Department of Environmental Quality From: Trevor Selch Date: // RE: Selenium Concentrations in Lake Koocanusa Resident Fish The following assessment details the results of sampling efforts conducted to determine the concentrations of selenium (Se) bioaccumulated in the resident fish population in Lake Koocanusa in response to upstream coal mine expansions in British Columbia (BC), Canada. Comparison of muscle tissue Se concentrations within a -year period (8-) confirm significantly increased concentrations that are approaching literature-proposed threshold levels and surpass British Columbia Ministry of Environment (BC MOE) tissue-based guidelines, and in turn suggest Se loading in the reservoir. The results of the analysis are presented below.. Introduction: Source of Selenium exposure for Lake Koocanusa Fish The mobilization of selenium (Se) released from expansive coal mines in the Elk Valley of southeastern BC have resulted in elevated concentrations in fish in downstream lotic systems (McDonald and Strosher 998; Golder Associates Ltd 8). Lentic systems are not present in the basin, however, until the Elk River flows through Elko Dam and enters Lake Koocanusa, just north of the BC-Montana border, making the reservoir a likely source for Se loading. Lake Koocanusa is considered to be sensitive to Se accumulation because physicochemical conditions exist for inorganic Se to be converted into toxic organic Se and accumulate in biota (Orr et al. ). Selenium concentrations in the water column of Lake Koocanusa have been increasing over the past decade (McDonald, 9). The Montana Department of Environmental Quality (DEQ) listed Lake Koocanusa as a threatened water body in its April, edition of the State's Integrated Water Quality Report.. Selenium Toxicity to Fish Described as the paradox of Se in fish, Se is nutritionally required in small quantities, yet becomes highly toxic in slightly greater amounts (Lemly 99; Lemly 999). The most sensitive biological end point is eggs, where newly hatched larval fish can experience teratogenic deformities and death while feeding off the Se-rich yolk sac, while adult fish still appear healthy (Lemly 99; McDonald and Strosher

2 998; Rudolph et al. 8). This atypical relationship could shift a fish population from un-impacted to complete reproductive failure with only a small increase in Se loading (Lemly 999).. Target Tissues and Tissue-based Threshold Criteria Several tissues in fish have been targeted for Se monitoring and evaluation of the biological effects, including whole body, muscle, liver, and eggs (Lemly 99). Gravid ovaries or eggs are the most sensitive biological endpoint, and are considered the most accurate measure of Se exposure since they integrate waterborne and dietary pathways (Lemly 99); however Se concentrations are not representative in immature gonads (Lemly 98; Gillespie et al. 988) or during non-spawning seasons (Sager and Cofield 98), making the egg collection challenging and often impractical. Moreover, obtaining gravid females from multiple species with diverse seasonal reproductive strategies adds further difficultly to using this tissue. Whole-body Se concentrations have been proposed for use as a national tissue-based criterion for Se (Hamilton, ; EPA, ), however the ability to process (grind/composite) high numbers of large fish (especially salmonids) for this analysis is often impractical. Therefore, collecting fish skeletal muscle tissue (fillets) samples is considered by Montana Fish, Wildlife and Parks (MFWP) the most practical approach to monitoring Se accumulation in multiple fish species. Biological effects thresholds for freshwater and anadromous fish tissues have been proposed (Lemly 99; Hamilton, ) for whole body ( µg/g dw), skeletal muscle (8 µg/g dw), liver ( µg/g dw), and ovaries and eggs ( µg/g dw). The BC MOE recently released fish tissue-based guidelines for selenium for whole-body and muscle tissues ( µg/g dw) and eggs ( µg/g dw; BC MOE ) and EPA has recently released External Peer Review Draft Aquatic Life Criteria for Selenium that propose., 8. and.8 ug/g dw for eggs, whole body, and fish muscle (EPA ). For the purpose of this study, Se concentrations in skeletal muscle tissue will be compared to literature biological effects thresholds and BC MOE guidelines to determine if Se concentrations in fish tissues are at levels of concern for Se toxicity.. Baseline Data Lake Koocanusa holds a very diverse fish community. The fish population has been monitored since 97 and was in flux for several decades after the creation of Libby Dam while the waterbody transitioned between a lotic and lentic system; before stabilizing more in recent years (Dunnigan et al. ). Samples of Se concentrations in various fish species had been collected by Montana Fish Wildlife & Parks (MFWP) in 8 as part of a transboundary fisheries investigation. Seven species of fish were sampled and analyzed for Se in muscle, liver, and sometimes gonad tissues (Deleray et al. ). Species included bull trout (threatened species), kokanee, longnose suckers, mountain whitefish, northern pikeminnow, peamouth, and rainbow trout. Not all species had all three tissues analyzed. Muscle tissue Se concentrations (µg/g dw) from Lake Koocanusa ranged from.7-. in peamouth,.-.9 in northern pike minnow,.-. in longnose suckers,.-. in kokanee, and.-. in bull trout (Deleray et al. ).. Inter-agency Monitoring Efforts During, monitoring staff from Montana Department of Environmental Quality (MDEQ) and biologists from MFWP began a coordinated planning effort to monitor Lake Koocanusa. The primary objective for Lake Koocanusa monitoring was to collect benchmarked data to inform a baseline study that will be the initiation event for annual status and trends monitoring events. A secondary

3 objective was to collect spatially and temporally robust data that may be used for trends analysis or to evaluate and predict fate, transport, and geochemical cycling of Se within the Lake. Sources of pollutants (particularly Se) to Lake Koocanusa are primarily upstream in British Columbia. Relative contribution assumptions are based on current knowledge of loading (McDonald, 9) and recent data collections. It is not known how waterborne and sediment transport of Se to Lake Koocanusa relates to recent observations of elevated Se concentration in biota. Once in the lake, pollutants remain in the water column and are transported through diffusion, are taken up by biota, are deposited in sediment, or possibly volatilized.. Methods Collections of fish for Se analysis coincided, in part, with annual sampling conducted by MFWP at longterm monitoring sites. Spring sampling consisted of sinking gill-nets sets at two sites, while fall sampling consisted of 7 sinking gill-nets at two sites. In addition, baited hoop-nets were deployed throughout the winter (December through April, and ) to sample burbot. A Sampling and Analysis Plan (SAP) was developed for the spring and fall fish-tissue sampling that details the monitoring locations and activities. In sampling, fish sizes were sorted and selected to replicate the same sized fish that were analyzed in 8, except for bull trout where all available fish were analyzed, and burbot, which were not collected in 8 sampling. Figure. Location of spring and fall fish sampling sites.

4 . Results Spring sampling at the Rexford and McGillvray sites occurred in May,. Fall sampling at the Canada and Rexford sites occurred in September,. In total, between spring and fall sampling events, fish were collected and analyzed for Se in muscle and ovaries (when present). Fish samples were analyzed for differences in fish length. Only peamouth were significantly different in length between years, and comparisons were analyzed using ANCOVA, with fish length as covariate. Comparison of fish Se concentrations using a t-test or ANCOVA between 8 and yielded significantly higher Se concentrations in for all fish species sampled (Figure, p<.), and resulted in mean increases of -7% in Se muscle tissue burdens in just five-years. The transboundary sampling effort conducted in Lake Koocanusa in 8 included fish collected in May, 8 at the Rexford site, except kokanee which were collected in the fall (Deleray et al. ). Significant differences between years were found by comparing just the Rexford sites between 8 and, and by pooling all the data between sites in ; therefore, data displayed shows the results of pooled sites. Longnose suckers contained the highest mean muscle Se concentration and had the largest increase in Se concentrations between years. Northern pike minnows had the lowest concentrations of all species analyzed in, but were still significantly higher than concentrations found in 8. Mean Se concentration measured in longnose suckers in exceeded the BC MOE guideline value of µg/g, while westslope cutthroat averaged just below this level (Figure ). 9 8 Seleinum conc. (ug/g dw.) 7 * * * * * * 8 BULL LNSU NPM KOK PEA RBT WCT Figure. Lake Koocanusa mean fish muscle tissue selenium (Se) concentrations µg/g dry weight (dw.) sampled in 8 (black bars) and (white bars) from all sites for bull trout (BULL), longnose suckers (LNSU), northern pikeminnow (NPM), kokanee (KOK), peamouth (PEA), rainbow trout (RBT), and westslope cutthroat trout (WCT). Selenium concentrations significantly different between years are labeled with an * (p<.). Error bars represent ± S.E. Hashed line (8 µg/g dw) represents literature based biological effects thresholds for selenium toxicity in skeletal muscle tissue. Solid line ( µg/g dw) represents BC MOE muscle tissue-based guideline.

5 Individual fish Se concentrations in muscle tissue were also plotted to compare with toxicological threshold values (8 µg/g dw.) and the BC MOE muscle tissue guideline value ( µg/g dw.). Several longnose suckers and westslope cutthroat trout had Se concentrations that approach the threshold level of concern, and two peamouth samples exceeded this mark (Figure ). Nearly all individual longnose sucker Se concentrations exceed the BC MOE muscle tissue guideline, while Se concentrations in peamouth, rainbow and westslope cutthroat trout frequently exceeded the guideline level. 9 Selenium conc. (ug/g dw.) 8 7 BULL KOK LNSU NPM PEA RBT WCT Figure. Lake Koocanusa individual fish muscle tissue selenium (Se) concentrations µg/g dry weight (dw.) sampled in from bull trout (BULL), longnose suckers (LNSU), northern pikeminnow (NPM), kokanee (KOK), peamouth (PEA), rainbow trout (RBT), and westslope cutthroat trout (WCT). Hashed line (8 µg/g dw) represents literature based biological effects thresholds for selenium toxicity in skeletal muscle tissue. Solid line ( µg/g dw) represents BC MOE muscle tissue-based guideline. Comparisons of Se concentrations found in fish muscle tissue among the three sampling sites showed very little site differences suggestive of spatial variability (Figure ). The largest difference between sites was found in peamouth where Se concentrations were much higher at the Rexford site compared to Canada and McGillvray. Northern pikeminnow Se concentrations were highest at the Canada site. Variability in Se concentrations among sites for all other species was low. Overall there was no distinct pattern between sampling site and fish Se concentrations.

6 9 Selenium conc. (ug/g dw.) 8 7 Canada Rexford McGillvray BULL LNSU NPM KOK PEA RBT WCT Figure. Lake Koocanusa mean fish muscle tissue selenium (Se) concentrations µg/g dry weight (dw.) sampled in from three sites (Canada, Rexford, and McGillvray) for bull trout (BULL), longnose suckers (LNSU), northern pikeminnow (NPM), kokanee (KOK), peamouth (PEA), rainbow trout (RBT), and westslope cutthroat trout (WCT; N=). Error bars represent ± S.E. Hashed line (8 µg/g dw) represents literature based biological effects thresholds for selenium toxicity in skeletal muscle tissue. Solid line ( µg/g dw) represents BC MOE muscle tissue-based guideline. Fish ovaries were obtained from gravid female fish from different species. In total, 7 ovary samples were analyzed for Se concentrations (Figure ). Peamouth exhibited the highest ovary Se concentrations, and several fish exceeded the Se ovary toxicological threshold value of µg/g dw., and the BC MOE guideline of µg/g dw. The only westslope cutthroat trout ovary sample collected also exceeded µg/g Se, but not the BC MOE guideline. Northern pikeminnow ovary samples and the single longnose sucker ovary sample were also approaching the biological effects threshold.

7 Selenium conc. (ug/g dw.) 8 8 KOK LNSU NPM PM RBT WCT Figure. Lake Koocanusa individual fish ovary tissue selenium (Se) concentrations µg/g dry weight (dw.) sampled in from kokanee (KOK), longnose suckers (LNSU), northern pikeminnow (NPM), peamouth (PEA), rainbow trout (RBT), and westslope cutthroat trout (WCT). Hashed line ( µg/g dw.) represents level of concern for selenium toxicity in ovary/egg tissue. Hashed line ( µg/g dw) represents literature based biological effects thresholds for selenium toxicity in ovary/egg tissue. Solid line ( µg/g dw) represents BC MOE egg tissue-based guideline. Non-lethal muscle-plug samples were collected from 78 burbot during the winters of and during scheduled hoop-net sampling efforts. Muscle-plug tissue samples were stored in a - C freezer for extended periods of time ranging from 8 to days. In, there was no significant relationship between Se concentration and hold time (Figure a). However, in, there was a significant positive relationship between hold time and muscle-plug Se concentration (Figure b). 7

8 Selenium conc. (ug/g) Selenium conc. (ug/g) Selenium conc. (ug/g) 7 Burbot muscle plugs R =., P=. D Graph Burbot muscle plugs R =..98, P<. Moyie Lake muscle plugs R =., P=.8 (a) (b) (c) 8 Days stored before analysis Figure. Regression plots of selenium (Se) concentrations from individual burbot muscle-plug samples collected in the winter of (a) and (b) and Moyie Lake (c) and stored frozen before analysis. Lab analysis was conducted on as-received samples. 8

9 Due to the limited tissue sample size of the muscle plugs, the analytical lab was not able to provide % moisture to correct individual samples for water loss, and only two samples reported % moisture, which were in great disparity. As a consequence, burbot muscle-plug samples collected from Lake Koocanusa were compared to a reference population from Moyie Lake (Figure c), south of Cranbrook, BC. Samples stored frozen for similar days from Lake Koocanusa and Moyie Lake were compared, and burbot samples from Lake Koocanusa had significantly higher Se concentrations than Moyie Lake (Figure 7; p<.). On average, burbot muscle-plugs Se concentrations collected from Lake Koocanusa were nearly times higher than reference fish from Moyie Lake. Selenium conc. (ug/g) Lake Koocanusa Moyie Lake Days stored before analysis Figure 7. Results from burbot muscle plug samples analyzed for selenium (Se) on as-received tissue between Lake Koocanusa and Moyie Lake from similar days stored frozen before analysis. Error bars for Se and days stored represent ± S.E.. Discussion The discovery of significantly higher Se concentrations in all fish species sampled between 8 and was surprising considering the relatively short period of time between sampling events. Unlike waterborne or sediment Se monitoring that is often highly variable both geographically and temporally, fish tissue sampling represents the best indicator of continuous Se exposure and bioavailability. Even so, McDonald (9) reported waterborne total Se concentrations in 8 to be averaging.8 µg/l, while Teck coal reported mean Se concentrations in the reservoir to be. µg/l in (Technical Advisory Committee Meeting No., Cranbrook, BC). Comparing increases in Se concentrations in water (7%) and all fish species muscle tissue (-7%) between 8 and suggests Se loading is occurring in Lake Koocanusa and should be of utmost concern for the long-term ecological health of the Lake Koocanusa fish community. 9

10 While the mean Se concentrations measured currently do exceed the recommended threshold levels, several species of fish exceed the BC MOE guidelines and results from analyses on individual fish exhibit Se concentrations are near, at, and above the proposed toxicological threshold levels. This finding is alarming and suggests there may already be reproductive failure effects occurring at the individual fish level from exposure to elevated Se concentrations accumulated in the tissues. This pattern is revealed again in the ovary Se results, where peamouth and Montana s State-fish, the westslope cutthroat trout, contain Se concentrations above threshold levels of concern. The fish species containing some of the highest Se concentrations include native fish, such as longnose suckers and peamouth. While not considered charismatic fauna by most anglers, these native species play a key role as pelagic and benthic planktivores and insectivores in an evolving and dynamic river system. Unlike highly bioaccumulative contaminants such as mercury, Se concentrations in fish tissues are often highest in small, lower-trophic-level fish. This phenomenon is attributable to high metabolic rates in younger fish (high energy needs) and food habits. While fish that feed on algae, zooplankton, and aquatic invertebrates are consuming prey items containing lower concentrations of Se, the lower energy content of the prey equates into much higher Se intake (Dubowy 989). For this reason, species like longnose suckers and peamouth are more susceptible toward accumulating high concentrations of Se in their tissues. Moreover, the habitat use of benthic species (i.e., longnose suckers and burbot) expose these species to the sediment-water interface where Se is likely settling and methylated by microbial activities. Only the Rexford site was sampled in both the spring and fall sampling events. Catch rates between spring and fall sampling events differ considerably because of differing life history strategies (i.e., spawning movements, seasonal habitat use) between species. Consequently, seasonal comparisons were not possible, however where species were caught in both the spring and fall, no differences in Se tissue concentrations were evident. Inter-annual variability in Se loading would be difficult to detect from fish tissues since the concentrations measured represent a wide temporal range of exposure. To capture this variability waterborne monitoring would be most representative. Beyond these species for which adequate tissue data exist, burbot are a species of concern in both Lake Koocanusa and the Kootenai drainage, and the population has been in decline since the mid-9s. Given their benthic habitat preferences, concerns over the potential for burbot to readily accumulate Se prompted a spontaneous muscle-plug collection effort to coincide with the winter hoop-netting season. Unfortunately, due to the remote location and logistics surrounding burbot muscle-plug collection over a -month period, samples were not obtained until the end of the season and were stored for long hold-times. Furthermore, when samples were submitted to the laboratory in to measure Se concentrations, a % moisture calculation was requested, however it later discovered that moisture content could not be measure due to the limited tissue content and laboratory processing methodologies. Consequently, to obtain an estimate of % moisture content in, three muscle-plug tissue samples were submitted separately for % moisture determination; however, the results from these samples were inconsistent and did not provide a reliable measure. Therefore, the concentrations represented by the burbot muscle-plugs are considered as-received for and, and are not wet weight or dry weight, but rather some indeterminate level. Still, by correcting for the holding times stored before the analysis, it is apparent that the concentrations in the Lake Koocanusa burbot are elevated compared to background fish obtained from Moyie Lake, a waterbody within the Kootenai drainage that is not influences by upstream mining.

11 In summary, the Se concentrations found in the resident fish in Lake Koocanusa are currently at levels of concern for ecological health. In five years, the concentrations have significantly increased in all species, from -7%. If the current trend of Se bioaccumulation continues, it may not be long before we witness the extirpation of fish year classes or even whole species from the system.. References BC MOE.. Companion Document to: Ambient Water Quality Guidelines for Selenium Update. Water Protection and Sustainability Branch. Environmental Sustainability and Strategic Policy Division. British Columbia Ministry of Environment. DeForest, D. K., K. V. Brix, and W. J. Adams Critical Review of proposed residue-based selenium toxicity thresholds for freshwater fish. Human and Ecological Risk Assessment. :87-8. Deleray, M, J. Cavigli, A. Steed, and T Selch.. Transboundary Flathead Fisheries Baseline Data Collection. Montana Fish, Wildlife & Parks. DuBowy, P. J Effects of diet on selenium bioaccumulation in marsh birds. J. Wildl. Manage. : Dunnigan, J., J. DeShazer, T. Ostrowski, M. Benner, J. Lampton, L. Garrow, and J. Tohtz.. Mitigation for the Construction and Operation of Libby Dam, 7//- // Annual Report. BPA Project Number EPA.. Draft Aquatic Life Water Quality Criteria for Selenium. EPA-8-D--. EPA.. External Peer Review Draft Aquatic Life Ambient Water Quality Criterion for Selenium Freshwater. EPA-HQ-OW--9. Gillespie, R. B., P. C. Baumann, and C. T. Singley Dietary exposure of bluegills (Lepomis macrochirus) to (7) Se: uptake and distribution in organs and tissues. Bull. Environ. Contam. Toxicol. : Golder Associates Ltd., 8. Selenium Status Report 7, Elk river Valley, BC. Report prepared for the Elk Valley Selenium Task Force. 7 pp. Hamilton, S. J.. Rationale for a tissue-based selenium criterion for aquatic life. Aquatic Toxicology. 7:8-. Hamilton, S. J. Review of residue-based selenium toxicity thresholds for freshwater fish. Ecotoxicology and Environmental Safety. :-. McDonald, L. E. and M. M. Strosher Selenium mobilization from surface coal mining in the Elk River Basin, British Columbia: A survey of water, sediment, and biota. BC Environment, Cranbrook, BC, Canada. McDonald, L.E.. Selenium Bioaccumulation Survey of Lake Koocanusa and Kinbasket Reservoir (). Env. Protect., B.C. Min. of Environ., Cranbrook, B.C. Dec. p.

12 McDonald, L.E. 9. Survey of selenium in water, zooplankton and fish in Lake Koocanusa, British C Columbia, 8. Env. Protect., B.C. Min. of Environ., Elk Valley Selenium Task Force., Cranbrook, B.C. Lemly, A. D. 99. Teratogenic effects of selenium in natural populations of freshwater fish. : 8-. Lemly, A.D. 99. Guidelines for evaluating selenium data from aquatic monitoring and assessment studies. Environ. Mon. and Assess. 8: 8-. Lemly, A.D Selenium impacts on fish: an insidious time bomb. Human and Ecological Risk Assessment. : 9-. Lemly, A. D Selenium transport and bioaccumulation in aquatic ecosystems: a proposal for water quality criteria based on hydrological units. Ecotoxicology and Environmental Safety. :-. Orr, P.L., K. R. Guiguer, and C. K. Russel.. Food chain transfer of selenium in lentic and lotic habitats of a western Canadian watershed: Ecotoxicology and Environmental Safety. : 8-9. Rudolph, B., I. Andreller, and C.J. Kennedy. 8. Reproductive success, early life stage development, and survival of westslope cutthroat trout (Oncorhynchus clarkii lewisi) exposed to elevated selenium in an area of active coal mining. Environ. Sci. Technol. (8): 9-. Sager, D.R., and C. R. Cofield. 98. Differential accumulation of selenium among axial muscle, reproductive and liver tissues of four warmwater fish species. Water Resour. Bull. :9-.