Arrow Reservoir Bull Trout

Arrow Reservoir Bull Trout:

Microchemical analysis of otoliths to determine stock structure, migration timing, and location of spawning and rearing habitats

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200 mol mol-1) mol μ

150 Sr:Ca ( Sr:Ca 100

0 0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 Distance (μm)

Prepared for:

James Baxter and Steve Arndt Fish & Wildlife Compensation Program 103-333 Victoria Street Nelson, BC V1L 4K3

Prepared by:

Adrian Clarke, M.Sc. and Kevin Telmer, Ph.D. Earthtone Environmental R&D Inc. 2675 Seaview Rd. Victoria, BC V8N 1K7 Executive Summary

A habitat alteration exists as a result of three dams which now influence Arrow Lakes Reservoir: Keenleyside Dam, Mica Dam, and Revelstoke Dam. These dams have permanently altered the pre-existing ecosystem and have impacted native fish populations such as bull trout. Critical information is needed to manage bull trout effectively including identifying natal spawning and rearing habitats, determining age of initial migration to the reservoir, and defining overall stock structure.

We determined the chemical composition of bull trout otoliths collected from the Arrow Lakes Reservoir (Nakusp creel survey), the Halfway River, Greeley Creek, McDonald Creek, Illecillewaet River, Incomappleaux River, Caribou Creek and Kellie Creek using Laser Ablation-Inductively Coupled Plasma Mass Spectrometry (LA-ICPMS). The goal of this study was to develop a model for Arrow Reservoir bull trout that determines stock structure, migration timing, and location of spawning and rearing habitats for both current and future otolith samples.

The results indicated that otolith chemistry analysis, through LA-ICPMS, is a useful technique for studying life history characteristics of fishes, specifically bull trout in the Arrow Lakes Reservoir. Strontium, Ba, Mg, Li and Mn provided symmetrical results throughout the otolith and the incorporation of these elements into the otoliths appears to be consistent over time. Most bull trout aged 1+ to 3+ years demonstrated significant movement among smaller tributaries in their larger rearing environment and reservoir entry occurred at approximately age 3+ for most of the individuals in this study. Validated classification accuracy using Discriminant Function analysis of juvenile bull trout was 94%. Seven out of nine unknown adult bull trout collected were classified to their natal tributaries while one individual was found to originate in the Arrow Lakes reservoir. This study was effective in demonstrating that microchemical analysis of fish otoliths combined with Discriminant Function analysis is an effective stock assessment tool for Arrow Lakes Reservoir bull trout. Acknowledgements

This project would not have been possible without the financial contribution of the Fish & Wildlife Compensation program and the technical expertise provided by Steve Arndt and James Baxter. In addition, Nicole Laforge at the School of Earth and Ocean Sciences, University of Victoria spent numerous hours preparing and analyzing the samples used in this study. Otolith collection was accomplished with the help of Glen Olson, Gail Olson, Jeff Burrows, John Hagen, and Jeremy Baxter. Finally, Scott Decker helped to collect the water samples for this project.

The Fish & Wildlife Compensation Program (FWCP) works on behalf of its Program Partners BC Hydro, the B.C. Ministry of Environment and Fisheries and Oceans Canada, to conserve and enhance fish and wildlife in the Canadian portion of the Columbia River Basin.

Table of Contents

Executive Summary...... 2 Acknowledgements...... 2 Table of Contents...... 3 List of Figures...... 3 List of Tables ...... 4 Introduction...... 5 Materials and Methods...... 6 Study Location...... 6 Water Analysis...... 8 Otolith Samples and Chemistry ...... 8 Statistical Analysis...... 9 Results and Discussion ...... 10 Water Chemistry ...... 10 Model Development...... 11 Life-History Profiles ...... 16 Conclusion ...... 20 Future Work...... 20 Literature Cited ...... 22 Appendix 1...... 24 Appendix 2...... 36

List of Figures

Figure 1. Overview of the Arrow Lakes Reservoir showing the tributaries included in the study (from Decker and Hagen 2007)...... 7

Figure 2. Sr:Ca and Ba:Ca values plotted (log10) for the Arrow Reservoir and each tributary sampled in 2007. Significant variation exists for each of the water-bodies examined...... 11

Figure 3. Classification of juvenile bull trout according to the results of the Discriminant function analysis with Sr:Ca, Ba:Ca, Mg:Ca, Mn:Ca, and Li:Ca as the predicting variables for the rearing period using measured elemental otolith concentrations. Adult bull trout (#57-#66) were classified as independent observations by the equation provided in the text. Overall classification success for the Discriminant model on juvenile bull trout was 96.9% and 93.8% when a jackknife analysis was completed...... 13

Figure 4. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #57...... 24

List of Tables

Table 2. Classification results for all locations sampled using Sr:Ca, Ba:Ca, Mg:Ca, Mn:Ca, and Li:Ca ratios to build the discriminant functions...... 14

Table 4. Functions at group centroids (mean values for each group) as determined by the discriminant model (Table 2, Figure 3)...... 15

Table 5. Age of the ten adult bull trout sampled from Arrow lakes Reservoir and the natal location classified to watershed. Each location was a new observation and evaluated using the classification coefficients developed by the Discriminant model. Each observation was classified using the following equation: (CF1)*Sr+(CF2)*Ba+(CF3)*Mg+(CF4)*Mn+(CF5)*Li-Constant = Discriminant score. Where CF refers to each coefficient determined by the discriminant function analysis. .16

Table 6. Estimated ages of all juvenile bull trout used in this study determined by Zn:Ca ratios...... 36

Introduction

Tracking fish through multiple life-history stages with conventional tagging techniques has contributed useful information to identify the timing and duration of habitat utilization. Additionally, stocks are usually defined by geographic separation and tagging information has been useful to discriminate between stocks. It has been determined, however, that there is an inherent bias in many tagging techniques as tagging programs often end up concentrating on the re-captured, non-mobile portion of the population (migrating fish often leave the study area), or on the members that are physically large enough to receive tags. Moreover, some species that are susceptible to injury from handling may experience higher mortality rates resulting directly from the application of physical tags. Partly, for these reasons, the amount of dispersal and timing of migration for various species and different age classes is poorly understood.

A method that shows promise for discriminating habitat use by freshwater fish is analyzing otoliths by laser ablation-inductively coupled plasma mass spectrometry (LA- ICPMS). Otoliths, which are calcified bony structures of the inner ear, function in the senses of balance and hearing (Popper et al. 2005). They are primarily composed of calcium carbonate (CaCO3) while K, Sr, Na, N, S, Cl, Ba, P, and other trace elements are the minor elements within the otolith (Campana 1999). Campana (1999) describes that even among the differences of elemental concentrations throughout the aquatic environment, specific trace elements, such as Sr, Zn, Pb, Mn, Fe, and Ba, within the otolith are consistently reflected in the environment. Because otoliths are metabolically inert, and permanently retain elements through daily growth, entire individual life histories can be recorded (Campana and Neilson 1985). The recorded life histories can then be analyzed through LA-ICPMS.

Laser Ablation-Inductively Coupled Plasma Mass Spectrometry is a technique that utilizes a narrow laser beam to scan the surface of a solid object (otolith). This technique has gained popularity because LA-ICPMS has the ability to analyze concentrations of single or multiple trace elements at high precision (Sanborn and Telmer 2003). Other benefits of this technique include easy sample preparation, few sample size limitations, and low probability of contamination (Sanborn and Telmer 2003). A number of studies have used LA-ICPMS in fisheries research, including stock discrimination and identification (Rooker et al. 2003), migratory and environmental history (Arai et al. 2007; Clarke et al. 2007a, b), age of fish based on seasonal variation in elemental signature of

5 the water (Clarke et al. 2007c), and fish physiology (Melancon et al. 2005; Arai and Hirata 2006).

In the present study we examined the elemental signatures from bull trout otoliths collected from the Arrow Lakes Reservoir (Nakusp creel survey), the Halfway River, Greeley Creek, McDonald Creek, Illecillewaet River, Incomappleaux River, Caribou Creek and Kellie Creek. The objective of this pilot study was to determine if LA-ICPMS will improve our understanding of early rearing life histories and migration timing for Arrow Reservoir bull trout. Specifically we aimed to develop a model for Arrow Reservoir bull trout that determines stock structure, migration timing, and location of spawning and rearing habitats for both current and future otolith samples. Once the model development is complete, we should also be able to determine the level of recruitment (stock strength) from known bull trout watersheds based on future analyses of adult bull trout collected in future creel surveys.

Materials and Methods

Study Location Arrow Lakes Reservoir is located in the West Kootenay region of British Columbia. The reservoir is 230 km long and was formed when the original Arrow Lakes and the Columbia River were impounded behind the Hugh Keenleyside Dam. The reservoir has several major tributaries (Figure 1).

Figure 1. Overview of the Arrow Lakes Reservoir showing the tributaries included in the study (from Decker and Hagen 2007).

7 Water Analysis Water analyses were completed independently by Maxxam Analytical Services, Burnaby British Columbia. All of the water samples were collected and preserved using syringe filters and HDPE collection bottles. All of the samples were acidified in the field using HNO3 according to instructions from Maxxam. Water samples were collected from several tributaries and locations in the reservoir in both 2006 and 2007. All of the water samples were collected from near the water surface except for those at stations AR2 and AR7 (upper Arrow Reservoir). At these two locations, one deep water sample was collected using a plastic Van Horne bottle (290m and 160m respectively), and one integrated sample was collected from 0-20m using a plastic tube with the collected water mixed in a plastic bucket. Six locations (Arrow Lakes, Illecillewaet River, Halfway River, McDonald Creek, Incomappleaux River and Caribou River were sampled in both years. Eleven locations in total were sampled, four in the reservoir and the remainder in the tributaries in 2007.

Otolith Samples and Chemistry Sagittal otoliths were collected from juvenile bull trout (n = 54) in the Halfway River, Greeley Creek, McDonald Creek, Illecillewaet River, Incomappleaux River, Caribou Creek and Kellie Creek. In addition 10 adult bull trout otoliths were collected in the Nakusp creel survey. The Nakusp Creel survey collected data and biological samples from anglers who were fishing in the Arrow Reservoir and using the Nakusp boat launch. It was assumed that adult bull trout collected in the survey were captured in the reservoir and not other lakes or rivers nearby.

Individual otoliths were stored in scale envelopes until processing. Otolith preparation was conducted by Earthtone Environmental R&D Inc. Samples were cleaned in deionized water, then embedded in epoxy (Buehler Epoxy-Cure Resin), which adds strength to the otolith preventing breakage. The otoliths, along with the epoxy covering, were then scored with a scalpel and sectioned using an isomet saw (Buehler). Secondary epoxy embedding was accompanied by placing the sectioned otolith into acrylic tubing where more epoxy was added to secure the otolith. The core was exposed by polishing the otolith with adhesive-backed lapping paper in 320, 600, and 1200 grit sizes (Buehler, Carbimet). To achieve a highly polished surface, otoliths were moistened with 0.25 µm diamond suspension spray (Buehler, Metadi Supreme) and polished with 2500 grit pads (Buehler, Texmet). LA-ICPMS was accomplished at the School of Earth and Ocean Sciences, University of Victoria, using the UP-213 Laser Ablation System (New Wave Research) attached to an X Series II ICP-MS (Thermo Electron Corporation). Concentrations of Sr, Ba, Mn, Ca, Mg, Li and Zn were identified in a transect line across the diameter of the otoliths. Prior to scanning, background data was collected for 20 seconds to separate the background signal from otolith elemental chemistry. Finally, PlasmaLab (version 2.5.3.280, Thermo Electron 2003) software was used for data collection and reduction.

8 Statistical Analysis The trace-metal chemistry in otoliths has been related to the trace-metal chemistry in water using an incorporation coefficient (Wells et al. 2003; Clarke et al. 2007). The coefficient is calculated as the molar ratio in the otolith over the molar ratio in the water (e.g. [Sr:Ca otolith ] / [Sr:Ca water ]). We calculated incorporation coefficients based on the water chemistry from the Arrow Reservoir and used 9 of the 10 adult bull trout otoliths from fish known to be in the Arrow Reservoir at time of capture; values of 0.296 and 0.0155 were calculated for Sr, and Ba, respectively. One bull trout was excluded from the dataset because measured Sr:Ca and Ba:Ca elemental ratios did not correspond to any known water chemistries in this study. The incorporation coefficients determined for bull trout in our study differed slightly from those previously reported by Clarke et al. (2007a) for Arctic grayling; 0.346 and 0.0484 for Sr and Ba, respectively but Sr:Ca was close to the value of 0.25 reported by Taylor and Clarke (2007) for bull trout. The incorporation coefficients were used to calculate the expected otolith elemental concentrations based on measured water chemistries for each tributary examined.

Discriminant function analysis with jack-knife resampling was used to provide a visualization of geographical separation using juvenile otoliths collected from the Halfway River, Greeley Creek, McDonald Creek, Illecillewaet River, Incomappleaux River, Caribou Creek and Kellie Creek using a combination of Sr:Ca, Ba:Ca, Mg:Ca, Li:Ca and Mn:Ca (SPSS version 12). Jack-knife re-sampling validates the robustness of the discriminant functions. The jack-knife approach is appropriate when sample sizes are too small to allow for a split sample procedure (Tabachnick & Fidell 2001). All of the adult bull trout were then incorporated into the model as new observations using the following equation (effectively a split sample procedure):

(CF1)*Sr+(CF2)*Ba+(CF3)*Mg+(CF4)*Mn+(CF5)*Li-Constant = Discriminant score

Where CF refers to each coefficient determined by the discriminant function analysis. The adult bull trout were then classified into their stream of origin based on their discriminant score.

Individual life-histories for each adult bull trout were examined using predicted locations from the discriminant model and expected Sr:Ca ratios determined via the incorporation coefficient. The discriminant model was built (predicted locations) using sections of the laser transect across the juvenile bull trout otolith that represented the period when each fish was in its rearing tributary. Adult bull trout were then classified to their predicted locations by using the same section of the otolith that was used in the juvenile model development (rearing tributary signal), but the equation above was used instead of discriminant function analysis using SPSS. Strontium is the main element in predicting location, and estimates of migration or rearing patterns can be accomplished effectively using this element. The use of the incorporation coefficient to determine expected Sr:Ca ratios in the otolith for each tributary combined with known Sr:Ca ratios (measured in juvenile otoliths for each tributary) helped us to determine individual life-histories for adult fish. In addition, age was estimated with Zn:Ca oscillations for all bull trout

9 examined in this study and estimated ages were used to determine timing of reservoir entry.

Results and Discussion

Water Chemistry All of the watersheds examined exhibited large differences in water chemistry (Table 1, Figure 2). There were also substantial differences in water chemistry within each individual watershed for tributaries examined. Water chemistry was not statistically evaluated due to inconsistencies between measured values in 2006 and 2007 for the same sampling location. The inconsistency in water chemistry appears to stem from the 2006 sampling year. Replicate samples (e.g. Illecillewaet River 2006) show considerable variation in measured water chemistries for water sampled at the same location on the same date. It appears the precision of the water analysis was compromised for 2006. Another possibility is that water chemistries fluctuate on an annual or seasonal basis in tributaries to the Arrow Reservoir. This conclusion seems unlikely as many of the otoliths examined suggested longer-term periods of consistency and replicate water samples are not identical. In addition, the spatial differences in the chemistry of freshwaters largely reflect differences in age and composition of the underlying bedrock. These differences result in variation among stream chemistries, but also provide a stable chemical signature for each river system. Consistency within river systems is well described by Taylor & Hamilton (1994) who examined 25 years of water chemistry data on the Saskatchewan River system and showed that element ratios remain consistent over time. Further support for the consistency in water chemistry is the stability of Sr, Ba and Mn measured across otoliths of slimy sculpins from the Williston watershed, British Columbia (Clarke et al. 2004a). Slimy sculpins are considered to be nonmigratory and the elemental concentrations maintained a flat profile for 2–5 years depending on the age of the sculpin. The lack of changes in elemental signature across much of the otolith indicates that chemical signatures have been stable for at least 5 years for the tributaries that were sampled. For these reasons it seems likely that water chemistries are also stable in tributaries of the Arrow Reservoir.

10 100

ILLECILLAWET R #1 GREELY C #1 INCOMAPLEUX R #1

10 CARIBOU C #1 KUSKANAX R #1 MACDONALD C #1 HALFWAY R #1 Reservoir

Ba:Ca (mmol-mol) Ba:Ca 1 10 100 1000

0.1 Sr:Ca (mmol-mol)

Figure 2. Sr:Ca and Ba:Ca values plotted (log10) for the Arrow Reservoir and each tributary sampled in 2007. Significant variation exists for each of the water-bodies examined.

Model Development The incorporation coefficients calculated for bull trout collected from the Nakusp Creel survey were used to estimate the location of those fish according to expected Sr:Ca (mmol-mol) and Ba:Ca (mmol-mol) concentrations that would be present in bull trout otoliths in the watersheds where water sampling was completed (Table 1). The incorporation coefficients generated were similar to other studies (Wells et al. 2003; Clarke et al. 2007a,b; Taylor and Clarke 2007) when water analyzed in 2007 was used to build the model. This suggests that measured water chemistries completed in 2007 are likely close to actual values; however, new samples should be collected and analyzed due to the discrepancies between sampling years. The expected otolith chemistry as determined by the incorporation coefficient and the 2007 water chemistry was used to estimate the natal rearing environment of each unknown sample in addition to the predicted location provided by the discriminant model.

Table 1. Expected otolith elemental concentrations based on measured water chemistry for tributaries of the Arrow Reservoir. Two tributaries, the Halfway River and the Kuskanax River, have measured Sr:Ca concentrations at least 5x higher than what is found in the Pacific Ocean. Values are provided for both sampling years and show the discrepancy between the two independent water analyses conducted for the two-years of data. 2006 samples were collected from September 16 to November 3, while 2007 samples were collected in July. Waterbody Water Chemistry 2006 Water Chemistry 2007 Expected Otolith Chemistry 2007 Sr:Ca Ba:Ca Sr:Ca Ba:Ca Sr:Ca Ba:Ca ILLECILLAWET R #1 6.7 1.3 3.6 1.3 427.7 7.9 ILLECILLAWET R #2 4.4 1.9 3.6 1.3 430.1 7.8 GREELY C #1 na na 2.6 0.6 306.9 4.0 GREELY C #2 na na 2.6 0.8 304.7 4.9 INCOMAPLEUX R #1 6.3 0.8 4.9 0.6 581.5 4.0 INCOMAPLEUX R #2 6.2 0.8 5.0 0.7 592.5 4.1 CARIBOU C #1 4.8 1.2 4.7 0.8 560.8 5.2 CARIBOU C #2 4.7 0.6 4.7 0.8 556.0 5.2 KUSKANAX R #1 na na 121.2 15.4 14364.4 95.7 KUSKANAX R #2 na na 120.8 15.0 14310.5 93.1 MACDONALD C #1 6.6 0.3 5.9 0.5 696.1 2.9 MACDONALD C #2 6.5 0.3 5.8 0.4 688.7 2.2 HALFWAY R #1 75.0 7.7 92.8 11.8 10991.8 73.0 HALFWAY R #2 72.2 7.4 93.2 11.7 11049.0 72.6 AL NARROWS #1 6.4 0.7 6.1 0.9 726.3 5.6 AL NARROWS #2 6.3 0.7 6.1 0.9 723.3 5.6 AL (AR7 0-20m #1) na na 6.1 0.9 726.3 5.6 AL (AR7 0-20m #2) na na 6.1 0.9 718.4 5.4 AL (AR7 160m #1) na na 6.3 0.8 740.6 5.2 AL (AR7 160m #2) na na 6.1 0.8 726.9 5.1 AL (AR2 0-20m #1) na na 5.3 0.9 632.5 5.4 AL (AR2 0-20m #2) na na 5.2 0.8 620.3 5.0 AL (AR2 290m #1) na na 6.1 0.8 727.0 5.1 AL (AR2 290m #2) na na 6.2 0.8 732.7 5.1

Discriminant function analysis using jack-knife resampling for the multivariate combination of Sr, Ba Mg, Mn, and Li over the entire juvenile life of the bull trout used the linear combination of the predictor variables to produce DCF1 and DCF2 and revealed good separation between bull trout populations (Fig. 3). Future water analysis should include Li as it appears to be a very strong predictor of location in the Arrow watershed. The percentage of correct classification for each group of bull trout is provided in Table 2. The total number of cases classified correctly by the discriminant model was 97% and with jack-knife validation was 94%.

12 Halfway 6 Caribou 5 McDonald 4 Incomappleaux

3 Kellie Illecillewaet 2 58 59 Greeley 1 Nakusp Creel 61 0 6057 66 63 57 58 -1 6462 criminant Function 2 2 Function criminant 59 -2 Dis 60 -3 61 -4 62 -5 63 -20-15-10-50 5 101520253064 Discriminant Function 1 66

13 Table 2. Classification results for all locations sampled using Sr:Ca, Ba:Ca, Mg:Ca, Mn:Ca, and Li:Ca ratios to build the discriminant functions. Location Predicted Group Membership Total Caribou Greeley Halfway Illecillewaet Incomappleaux Kellie McDonald Nakusp creel Caribou Original Count Caribou 11 0 0 0 0 0 0 0 11 Greeley Ck 0 5 0 0 0 0 0 0 5 Halfway 0 0 12 0 0 0 0 0 12 Illecillewaet 0 0 0 7 0 0 0 0 7 Incomappleaux 0 0 0 0 7 0 0 0 7 Kellie Ck 0 0 0 0 0 2 1 0 3 McDonald 0 0 0 0 0 0 10 0 10 Nakusp creel 1 0 0 0 0 0 0 8 9 % Caribou 100.0 .0 .0 .0 .0 .0 .0 .0 100.0 Greeley Ck .0 100.0 .0 .0 .0 .0 .0 .0 100.0 Halfway .0 .0 100.0 .0 .0 .0 .0 .0 100.0 Illecillewaet .0 .0 .0 100.0 .0 .0 .0 .0 100.0 Incomappleaux .0 .0 .0 .0 100.0 .0 .0 .0 100.0 Kellie Ck .0 .0 .0 .0 .0 66.7 33.3 .0 100.0 McDonald .0 .0 .0 .0 .0 .0 100.0 .0 100.0 Nakusp creel 11.1 .0 .0 .0 .0 .0 .0 88.9 100.0 Cross- Count Caribou 11 0 0 0 0 0 0 0 11 validated(a) Greeley Ck 0 5 0 0 0 0 0 0 5 Halfway 0 0 12 0 0 0 0 0 12 Illecillewaet 0 0 0 7 0 0 0 0 7 Incomappleaux 0 0 0 0 7 0 0 0 7 Kellie Ck 0 0 0 1 0 0 2 0 3 McDonald 0 0 0 0 0 0 10 0 10 Nakusp creel 1 0 0 0 0 0 0 8 9 % Caribou 100.0 .0 .0 .0 .0 .0 .0 .0 100.0 Greeley Ck .0 100.0 .0 .0 .0 .0 .0 .0 100.0 Halfway .0 .0 100.0 .0 .0 .0 .0 .0 100.0 Illecillewaet .0 .0 .0 100.0 .0 .0 .0 .0 100.0 Incomappleaux .0 .0 .0 .0 100.0 .0 .0 .0 100.0 Kellie Ck .0 .0 .0 33.3 .0 .0 66.7 .0 100.0 McDonald .0 .0 .0 .0 .0 .0 100.0 .0 100.0 Nakusp creel 11.1 .0 .0 .0 .0 .0 .0 88.9 100.0 survey a Cross validation (Jacknife re-sampling) is done only for those cases in the analysis. In cross validation, each case is classified by the functions derived from all.

14 All of the adult bull trout obtained from the Nakusp creel survey were examined independently using the discriminant model developed using juvenile bull trout. The coefficients in Table 3 were used in combination with measured Sr:Ca, Ba:Ca, Mg:Ca, Mn:Ca and Li:Ca ratios to infer the natal habitat of each fish. Table 4 provides the mean value for each classified group according to each of the functions derived from the discriminant model.

Table 3. Canonical discriminant function classification coefficients from juvenile bull trout collected at known locations used to classify new observations (adult bull trout natal life-history). Once the model is developed all samples are run as unknowns to determine classified location. Function 1 2 3 4 5 ln SR 10.809 .852 -1.488 -.487 -.288 ln Ba -.367 -.746 2.492 .454 .134 ln Mg .518 -.318 .601 2.604 -.260 ln Mn .001 .343 -.346 .114 .757 ln Li -.178 3.204 -.827 -.297 -.800 (Constant) -78.004 -5.236 2.817 -9.523 2.260

Table 4. Functions at group centroids (mean values for each group) as determined by the discriminant model (Table 2, Figure 3). Location Function 1 2 3 4 5 Caribou -5.001 -1.739 .895 1.491 .075 Greeley Ck -13.442 .773 .377 -.345 -.249 Halfway 24.043 .187 .185 -.024 -.096 Illecillewaet -9.521 2.479 .532 -.034 -.112 Incomappleaux -2.191 3.102 -1.028 .601 -.147 Kellie Ck -2.707 .603 2.950 -1.991 1.039 McDonald -3.040 -1.061 -1.640 -.310 .745 Nakusp creel survey -5.088 -1.916 -.325 -1.031 -.799

Seven of the ten adult bull trout examined were classified into their natal habitat using the model developed (Table 5). Two of the fish had characteristic elemental values that matched the Arrow Reservoir at the time of capture but could not be classified by the model. Further refinement of the model by examining all of the major bull trout spawning watersheds and the inclusion of more 0+ to 1+ aged samples should allow for a higher classification success for unknown samples. One bull trout (#65) was very different from all other fish examined and was captured in an area where water chemistry is presently unknown. This fish was not included in the discriminant model.

Zinc:Calcium ratios were used to estimate the ages of bull trout in the study and determine age of reservoir entry. Halden et al. (2000) determined that seasonal deposition of Zn in Arctic char otoliths correlates to annulus formation. Milner (1982), as well as Bradley and Sprague (1985), suggest that metabolic rate influences Zn deposition in fish otoliths. Seasonal summer temperatures likely positively influence the uptake and production of Zn, while colder winter temperatures would represent times when lower levels of Zn uptake occur (Halden et al. 2000). Clarke et al. (2004b) also determined that oscillations of Zn present in fin-rays of

15 bull trout represent yearly increments because ages estimated using Zn:Ca ratios corresponded well to independent age estimates provided by North/South Consulting who used traditional ageing techniques. Halden et al. (2000) noted that the incorporation of Zn into Arctic char otoliths decreases with age. The results of Halden et al. (2000) are also consistent with other studies examining Zn uptake by fish (Milner 1982; Bradley and Sprague 1985; Campbell and Stokes 1985). Our results also suggest that Zn:Ca oscillations are very strong up to age 4, and are more difficult to define as bull trout get older. Adult bull trout ages according to Zn:Ca ratios are provided in Table 5 while the ages determined for the 54 juvenile bull trout are provided in Appendix 1.

Table 5. Age of the ten adult bull trout sampled from Arrow lakes Reservoir and the natal location classified to watershed. Each location was a new observation and evaluated using the classification coefficients developed by the Discriminant model. Each observation was classified using the following equation: (CF1)*Sr+(CF2)*Ba+(CF3)*Mg+(CF4)*Mn+(CF5)*Li- Constant = Discriminant score. Where CF refers to each coefficient determined by the discriminant function analysis. Adult Fish Number Length (mm) Zn Age (years) Natal stream classification

57 630 6 or 7 Greeley

58 750 6 Illecillewaet

59 630 6 Incomappleaux

60 580 6 Greeley

61 660 6 Not Classified

62 730 7 Caribou

63 540 6 Halfway

64 760 7 or 8 Reservoir

65 640 7 Unknown capture location

66 800 Unknown Caribou

Life-History Profiles Life-History profiles were developed for each of the 10 adult bull trout using the natal location determined from the discriminant model, measured Sr:Ca ratios across the diameter of the otolith, and Zn:Ca oscillations to provide an estimate of age. The first fish examined (bull trout #57) entered the reservoir at age 3+ according to Zn:Ca oscillations and the sharp increase in Sr:Ca ratios at approximately 2300 um on the x-axis (Figure 4, Appendix 1). The Sr:Ca profile suggests that this fish has not returned to its natal tributary (Greeley Creek) to spawn and has spent its entire adult life-history in an environment with elemental concentrations that match the Arrow Reservoir (where it was captured). Zn:Ca oscillations are difficult to interpret after age 3+ for bull trout #57 but it appears that this fish was six or possibly seven years old at the time of capture. The decreasing oscillations of Zn found in all adult bull trout in this study are likely due to: (a) relative homogeneity of the reservoir; (b) larger fish have greater stores and greater residence time of elements so the overall chemistry of larger fish changes more slowly; and (c) fish otoliths grow in 3 dimensions resulting in a thinner layer each

16 year (with greater mass) available for ablation. Growth of bull trout #57 accelerated after reservoir entry between the ages of 3+ and 4+ and was likely due to an increase in the availability of larger prey species. The acceleration in growth observed in bull trout #57, and all other adult bull trout in this study when the fish entered the reservoir, is much greater than what Figure 4, and the following figures in Appendix 1 depict. Distance between annuli is not a perfect measure of growth as an otolith is a 3 dimensional object and so even if the same mass is added each year the thickness of layers will decrease roughly 4/3(π)r2^3(time 2) - 4/3(π)r1^3(time 1) which reduces to r2^3(time 2)-r1^3(time 1) if otoliths were spheres. For us to observe a sharp increase in growth using a linear scale suggests that growth of bull trout increases significantly when they enter the Arrow Lakes Reservoir.

Bull trout #58 reared in the Illecillewaet River until age 3 when it entered an environment (possibly the Columbia River at Revelstoke) with slightly higher Sr:Ca ratios than the Arrow Reservoir (Figure 5, Appendix 1). This fish then moved into the reservoir between age 3 and 4 where it likely resided for the rest of its life- history. The small fluctuations in Sr:Ca ratios after age 4 are likely indicative of movements within the reservoir but could also signify movements into tributaries of similar chemistry or feeding events at the outlet of tributaries. The reservoir may also be chemically stratified and the observed fluctuation could simply be movements between the strata. The water chemistry data collected for the reservoir did not suggest chemical stratification but most samples were collected in the northern arm of the Reservoir. There is no indication, according to Sr:Ca ratios, that bull trout #58 had returned to the Illecillewaet River. Bull trout #58 appears to be 6 years of age, however one more Zn:Ca oscillation may be present between 3 and 4. If this fish is 7 years of age, reservoir entry occurred after age 4, resulting in an increased growth rate between age 4 and 5.

Bull trout #59 provides an example of the suspect water chemistry data provided for this report (Figure 6, Appendix 1). The Sr:Ca values expected for Incomappleaux River bull trout according to the juvenile bull trout analyzed (captured in Incomappleaux River) are ~950 (mmol-mol-1) while expected values according to the juvenile bull trout captured in Kellie Creek are 1000-1200 (mmol-mol-1). Measured water chemistries in the Incomappleaux River result in a much lower Sr:Ca expected otolith concentration for Incomappleaux River bull trout (Table 1). An alternate hypothesis for the differences observed in measured water Sr:Ca and measured otolith Sr:Ca is that fish captured in the Incomappleaux River were not from the same location as the water sampling. The model built using juvenile otoliths suggests that bull trout #59 moved between Kellie Creek and the Incomappleaux River until age 4 when it migrated into the Arrow Reservoir. The accelerated growth of bull trout #59 also coincides with the inferred movement into the reservoir according to measured Sr:Ca ratios in the otolith. Bull trout # 59 never moved back into its natal habitat after reservoir entry according to measured Sr:Ca ratios in the otolith. One weakness with the use of LA-ICPMS is that we cannot observe the last month of a fish’s life. Although the 10 um resolution for the laser used to ablate these otoliths corresponds to c.1 week of otolith growth, shorter term signals can still be detected; the magnitude of the change in concentration is less for short-term changes due to target mixing, but it is still clearly observable. This is known from the analysis of otoliths from chemical tagging experiments where fish are exposed to elevated Sr concentrations for just a few hours. In such analysis, a beam resolution of 50 um was able to clearly detect exposures of just 6 h (Telmer et al., 2006). Material deposited onto the otolith just before death, however, is difficult to analyse because it is right at the edge of the target, and so a short end-of-life freshwater signal by the nature of its location in the otolith is difficult to observe. Other techniques can be used to detect the chemical signal at the edge of otoliths in combination with LA-ICPMS if that end of life signal is desired.

The portion of the line scan on the right side of Figure 6 after age 0 is more condensed. The more condensed portion of the graph represents the same amount of time (i.e. age 0 to age 6), however the laser tracked over a

17 portion of the otolith where annuli were more tightly stacked. The imperfect symmetry of otoliths, particularly for metabolically controlled elements (e.g. Zn), that are more strongly uptaken in the protein matrix of otoliths, results in a higher concentration on one side of the otolith than the other (higher concentration of endolymphatic fluid). The more compressed side of the otolith is always lower in Zn. A good example of Zn concentration being higher on one side of the otolith can be examined in Figure 8, Appendix 1. Another interesting observation for bull trout #59 is that Zn:Ca and Sr:Ca appears correlated. Bull trout #59 appears to have made seasonal (summer vs. winter) movements where habitats have distinct Sr:Ca concentrations. These seasonal movements coincide with the change in metabolic uptake of Zn between the two seasons.

The discriminant model suggested that bull trout #60 originated in Greely Creek (Figure 7, Appendix 1). Bull trout #60 appears to have migrated from Greeley Creek at age 2 into the Illecillewaet River. This fish made one migration into the reservoir between age 3 and 4 before entering a habitat with Sr:Ca ratios exceeding 900 (mmol-mol-1). The fish then migrated back into the reservoir between age 4 and 5 (winter). The fish then appears to have returned to the habitat with high Sr:Ca ratios at age 5+ before moving back into the reservoir where it was captured in the summer at age 6.

Bull trout #61 appears to have entered the reservoir at age 3+ although it is difficult to distinguish whether or not this fish is age 6 or 7 using Zn:Ca oscillations (Figure 8, Appendix 1). There may be an additional peak between 3 and 4 which would suggest this fish moved into the reservoir at age 4. Growth also signifies reservoir entry at approximately 2500 um on the x-axis due to the accelerated pattern of growth observed in the otolith between annuli. Bull trout #61 was not classified by the discriminant model so movement patterns cannot be inferred.

Bull trout #62 was classified by the discriminant model as originating in Caribou Creek (Figure 9, Appendix 1). This fish made small migrations between Caribou Creek and another tributary until age 2+ when it moved into an environment with Sr:Ca ratios approaching 1100 (mmol-mol-1). Reservoir entry appears to have occurred between age 4 and 5, although smaller unknown movements are apparent. The unknown movements could be a combination of unknown tributary habitats (i.e. water chemistry) or mixed chemical environments (e.g. the outlet or confluence of two chemical habitat types).

Bull trout #63 remained in the Halfway River watershed until age 3+ when it moved into the Arrow Reservoir Figure 10, Appendix 1). Bull trout #63 appears to have made movements within the Halfway River between at least two chemical habitat types. An alternate explanation could be that the very high Sr:Ca ratios observed could fluctuate annually depending on the source of these unusually high Sr signals (e.g. hot spring output diluted by run-off). Further sampling of water chemistries within this environment could elucidate the cause of these fluctuations (i.e. fish movements or chemical changes due to source inputs). Bull trout #63 appears to have remained in the Arrow Reservoir until time of capture. Interestingly, Sr:Ca ratios decline slightly after entry into the reservoir but remain higher than Sr:Ca ratios measured in other bull trout captured in the reservoir. The most likely explanation is that mixing is occurring between the Halfway watershed and the Arrow Lakes Reservoir and Sr:Ca ratios are slightly higher in this portion of the reservoir due to the high input from the river (bull trout #63 resided for the most part near the confluence of the Halfway River and the Arrow Lakes Reservoir).

Bull trout #64 appears to have entered the reservoir at age 4 based on measured Sr:Ca ratios and the observed increase in growth rate (Figure 11, Appendix 1). The fish has 7 fairly clear oscillations of Zn:Ca, however one more oscillation between 5 and 6 may exist indicating that this fish is 8 years of age. Zn:Ca ratios were

18 only consistent for one side of the otolith. Bull trout #64 was classified as originating in the Arrow Reservoir (likely entered as young of the year), however the location of its plotted location on Figure 2 suggests it originated from an unknown habitat. Further refinement of the discriminant model, with the addition of samples from more watersheds, would likely permit correct classification of this fish and improve overall classification of future samples.

We did not include bull trout #65 in the discriminant model because the chemical habitat where this fish was captured was not at all similar to any measured chemical habitats in the watershed (Figure 12, Appendix 1). At age 2 bull trout #65 left its natal rearing habitat and entered the unknown habitat where it resided for approximately five years. Sr:Ca values were approximately 100 mmol-mol in the habitat bull trout #65 entered. The habitat where this fish was captured was very constant and showed very little fluctuation over the 4 years for Sr:Ca ratios suggesting this fish may have been restricted in its movement patterns (trapped?). None of the tributaries measured in this study or other bull trout had Sr:Ca values this low. It appears that this fish was captured in a tributary to the Arrow Reservoir or another waterbody not measured for water chemistry and then obtained through the Nakusp Creel survey.

Bull trout #66 exhibits a very unique life-history for this data-set (Figure 13, Appendix 1). The Sr:Ca profile remains relatively consistent for the entire scan. Further Zn:Ca does not show definite oscillations after the 1st year of life. Classification by the discriminant model suggests this fish reared in Caribou Creek but it is difficult to determine when reservoir entry occurred. The most likely life-history scenario for this individual is movements between the confluence of Caribou Creek and the reservoir but the similarity in the chemical environments experienced by bull trout #66 make any inference difficult. It may simply be that this fish was flushed out into the reservoir as a juvenile fish and has remained in the reservoir environment for its entire life-history. The small fluctuations in Zn:Ca also indicates that this fish has remained in a fairly homogenous environment.

The life-history observed for bull trout #3 captured in the Halfway watershed demonstrates a mobile juvenile bull trout (Figure 14, Appendix 1). This fish has made seasonal migrations between the Halfway River (Sr:Ca ~12000 mmol mol-1) and an unknown water-body between age 0 and 2 with a Sr:Ca ~ 4500 mmol-mol-1. One tributary measured in this study, the Kuskanax River, has expected otolith Sr:Ca ratios of 14000 mmol- mol-1 and may be the 3rd habitat bull trout #3 is using, however this is unlikely as the two watersheds are 25 km apart. Bull trout #3 is most likely using tributary habitat within the Halfway watershed with very high Sr:Ca ratios that are similar to the Kuskanax expected Sr:Ca values. It difficult to determine the actual movement patterns until a more comprehensive water sampling or juvenile otolith collection program takes place. After age 2 this bull trout has a very similar life-history to bull trout #63. The Zn:Ca pattern suggests that the fish is spending the winter in the water-body with the lower Sr:Ca values. For future model development (for classification of unknown adults) it would be useful to only include juvenile fish that do not demonstrate high mobility (see next figure). Bull trout #3 shows three distinct oscillations of Zn:Ca suggesting this fish is 3 years old.

Bull trout #26 provides an example of younger bull trout with a more restricted movement pattern during its short life-history (Figure 15, Appendix 1). Zn:Ca ratios suggest this bull trout is 2+ years old. The portion of the line scan used to build the model for determining location (unknown adult classification to natal watershed) would be between 0-150 um and 530-675 um on the x-axis. The core of the otolith is avoided as it does not necessarily represent the water chemistry of where the fish is rearing due to physiological (maternal incorporation) and geochemical (the core is mineralized differently than the rest of the otolith) mechanisms.

19 Conclusion

The results indicate that otolith chemistry analysis, through LA-ICPMS, is a useful technique for studying life history characteristics of fishes, specifically bull trout in the Arrow Reservoir. Strontium, Ba, Mg, Li and Mn provided symmetrical results throughout the otolith and the incorporation of these elements into the otoliths appears to be consistent over time. Previous work has shown that elemental signatures are directly proportional to water chemistry (Wells et al. 2003 for cutthroat trout; Clarke et al. 2004 for slimy sculpins; Clarke et al. 2007a for Arctic grayling). The age of entry into the reservoir appears to correspond well with other studies examining bull trout life-history. The majority of bull trout across their range rear in streams until the age of 3+ (McPhail and Baxter1996). We did observe significant movement among rearing tributaries for the juvenile bull trout analyzed in this study; particularly fish aged over 1+. The variation in concentrations of Sr and Ba from the LA-ICPMS of otoliths from the juvenile rearing phase did increase the variation for our estimates; however, the overall watershed variation still allowed classification to the watershed level at which water chemistries were measured. The results of this study could be more clearly refined with the use of younger age bull trout. It appears that bull trout aged 2-3 years demonstrate significant movement among smaller tributaries in their larger rearing environment. The simple interpretation of this finding is that the fish did not remain in the same habitat throughout their entire juvenile freshwater stage.

Future Work Our results for Arrow Reservoir bull trout indicate that LA-ICPMS is a valid technique for determination of life-history characteristics and behaviors of fishes in this watershed. The model as developed will be an effective stock assessment tool for predicting the natal watersheds of adult bull trout captured in the fishery. An original assumption of this work was that we could rely on the water chemistry data to build an effective model, without the use of juvenile otoliths. More water chemistry data should be collected for additional testing as the variability in the dataset between sampling years is very uncommon. The following are specific recommendations that will help refine the model and make microchemical analysis of bull trout otoliths an effective tool for understanding bull trout life-history and fishery impacts in the Arrow Lakes Reservoir:

¾ Collect additional water samples in duplicate at mainstem and tributary locations where known bull trout populations exist. Restrict water sampling initially to a small number of watersheds but complete an intensive sampling regime where both mainstem and tributary samples are collected in both July and October of the same year. It is recommended that this is done in one watershed with a strong hotspring influence (e.g. Halfway) and one watershed without (e.g. Caribou Creek). To collect water samples the use of 125 mL HDPE amber bottles and polyethylene/polypropylene 50-mL syringes combined with syringe tip filters (25 mm ×0.22 μm), make water sampling efficient and ensures the integrity of the sample. Water samples should be acidified with a solution containing 0.4% high purity nitric acid (v/v addition, and environmental grade HNO3) in the field immediately after collection. All water samples should be kept cool and dark until analysis. It is also recommended that field blanks are made by adding the same amount of acid used to preserve the samples into DI water, and then storing the blanks the same as the water samples. Water analysis should also include Lithium as this element was useful in predicting habitats for Arrow Reservoir bull trout using otoliths. ¾ Collect more young bull trout (age 0+ to 1+) to enhance the predictive power of the model and improve classification of unknown adult samples into their natal environments. There was one adult sample that was captured in an unknown environment (Nakusp Creel fish #65) and one adult sample we could not classify to its rearing environments. Additional juvenile samples could be used in conjunction with new water chemistry data once the values are confirmed.

20 ¾ Analyze new adult bull trout otoliths collected in the Nakusp Creel survey. Valuable insight into the contribution of recruited bull trout to the Arrow Lakes Reservoir from each producing tributary could be gained using microchemical analysis of otoliths. The model developed in the present study, with some minor refinement, will be a useful stock assessment tool to further our understanding of bull trout stock structure, life-history, and the effects the sport fishery has on the individual populations residing in the Arrow Lakes watershed.

21 Literature Cited

Arai, T. and Hirata, T. 2006. Determination of trace elements in otoliths of chum salmon Oncorhynchus keta by laser ablation-ICP-mass spectrometry. Fisheries Science 72: 977-984.

Arai, T., Hirata, T., and Takagi, Y. 2007. Application of laser ablation ICPMS to trace the environmental history of chum salmon Oncorhynchus keta. Marine Environmental Research 63: 55-66.

Bradley R.W., and Sprague J.B. 1985. The influence of pH,water hardness, and alkalinity on the acute lethality of zinc to rainbow trout (Salmo gairdneri). Canadian Journal of Fisheries and Aquatic Sciences 42: 731-736.

Campana, S.E. & Thorrold, S.R. 2001. Otoliths, increments, and elements: key to a comprehensive understanding of fish populations? Canadian Journal of Fisheries and Aquatic Sciences 58, 30-38.

Campana, S.E. 1999. Chemistry and composition of fish otoliths: pathways, mechanisms, and applications. Marine Ecology Progress Series 188: 263-297.

Campana, S.E. and Neilson, J.D. 1985. Microstructure of fish Otoliths. Canadian Journal of Fisheries and Aquatic Sciences 42: 1014-1032.

Campbell, P. C. G., and Stokes, P. 1985. Acidification and toxicity of metals to aquatic biota. Canadian Journal of Fisheries and Aquatic Sciences 42: 2034–3049.

Clarke, A.D., Telmer, K., and Shrimpton, J.M. 2004a. Discrimination of habitat use by slimy sculpin (Cottus cognatus) in tributaries of the Williston Reservoir using natural elemental signatures. Peace / Williston Fish and Wildlife Compensation Program Report No. 288. 33pp.

Clarke, A. D. 2004b. Elemental signatures in bone to determine life history characteristics in fish. MSc Thesis, University of Northern British Columbia.122pp.

Clarke, A.D., Telmer, K.H., and Shrimpton, J.M. 2007a. Habitat use and movement patterns for a fluvial species, the Arctic grayling, in a watershed impacted by a large reservoir: evidence from otolith microchemistry. Journal of Applied Ecology 44: 1156-1165.

Clarke, A.D., Telmer, K.H., and Shrimpton, J.M. 2007b. Elemental analysis of otoliths, fin rays, and scales: a comparison of bony structures to provide population and life-history information for the Arctic grayling (Thymallus arcticus). Ecology of Freshwater Fish 16: 354-361.

Clarke, A.D., Lewis, A., Telmer, K.H., and Shrimpton, J.M. 2007c. Life history and age at maturity of an anadromous smelt, the eulachon Thaleicthys pacificus. Journal of Fish Biology 71: 1479-1493.

Halden, N. M., S. R. Mejia, J. A. Babaluk, J. D. Reist, A. H. Kristofferson, J. L. Campbell & W. J. Teesdale. 2000. Oscillatory zinc distribution in Arctic char (Salvelinus alpinus) otoliths: The result of biology or environment? Fisheries Research 46: 289–298.

McPhail, J.D. and Baxter, J.S.1996. A Review of Bull Trout (Salvelinus confluentus) Life-history and Habitat Use in Relation to compensation and Improvement Opportunities. Department of Zoology, U.B.C. Fisheries Management Report No. 104. 31pp

Melancon, S., Fryer, B.J., Ludsin, S.A., Gagnon, J.E., and Yang, Z. 2005. Effects of crystal structure on the uptake of metals by lake trout (Salvelinus namaycush) otoliths. Canadian Journal of Fisheries and Aquatic Sciences 62: 2609-2619.

Milner, N. J. 1982. The accumulation of zinc by O-group plaice, Pleuronectes platessa (L.), from high concentrations in seawater and food. Journal of Fish Biology 21: 325 -336.

Pannella, G. 1971. Fish otoliths- daily growth layers and periodical patterns. Science 173, 1124.

Popper, A.N., Ramcharitar, J., and Campana, S.E. 2005. Why otoliths? Insights from inner ear physiology and fisheries biology. Marine and Freshwater Research 56: 497-504.

Rooker, J.R., Secor, D.H., Zdanowics, V.S., De Metrio, G., and Orsi Relini, C. 2003. Identification of Atlantic bluefin tuna (Thunnus thynnus) stocks from putative nurseries using otolith chemistry. Fisheries Oceanography 12: 1-10.

Sanborn, M. and Telmer, K. 2003. The spatial resolution of LA-ICP-MS line scans across heterogeneous materials such as fish otoliths and zoned minerals. Journal of Analytical Atomic Spectrometry 18: 1231-1237.

Tabachnick, B.G., and Fidell, L.S. 2001. Using Multivariate Statistics, 4th edn. Boston, Allyn and Bacon.

Taylor, B.R. and Hamilton, H.R. 1994. Comparison of methods for determination of total solutes in flowing waters. Journal of Hydrology 154: 291-300.

Taylor, E.B., and Clarke, A.D. 2007. Microsatellite DNA and otolith microchemistry analysis of populations of bull trout (Salvelinus confluentus) in tributaries to the upper Fraser River, British Columbia. University of British Columbia, 31 pp.

Telmer, K., Penney, Z., Hamilton, A., Hume, J., Lofthouse, D. & Sheng, M. 2006. Strontium concentration profiles in reared juvenile sockeye salmon otoliths. Transaction of the 13th Ocean Sciences Meeting, American Geophysical Union 87 (Supplement), Abstract 0S45J-12. Available at http://www.agu.org/cgi-bin/SFgate/SFgate

Wells, B.K., Rieman, B.E., Clayton, J.L., Horan, D.L., and Jones, C.M. 2003. Relationships between water, otolith, and scale chemistries of westslope cutthroat trout from the Coeur d’Alene River, Idaho: the potential application of hard-part chemistry to describe movements in freshwater. Transactions of the American Fisheries Society 132: 409-424.

23 Appendix 1

0 1 2 3 4 5 6 900 180.00

800 160.00

700 140.00

600 120.00

500 100.00

400 80.00

300 60.00 Zn:Ca (mmol mol-1) Zn:Ca (mmol Sr:Ca (mmol mol-1) Sr:Ca 200 40.00

100 20.00

0 0.00 0 500 1000 1500 2000 2500 3000 Distance (μm)

Figure 4. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #57.

24 0 1 2 3 4 5 6 1050 250.00

900 200.00 750

600 150.00

mol mol-1) mol 450 100.00 300

50.00 Zn:Ca mol-1) (mmol

Sr:Ca (μ Sr:Ca 150

0 0.00 0 500 1000 1500 2000 2500 3000 3500 Distance (μm)

Figure 5. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #58.

25 6 5 4 3 2 1 0 1600 180.00

1400 160.00 140.00 1200 120.00 1000 100.00 800

mol mol-1) mol 80.00 μ 600 60.00

400 40.00 mol-1) Zn:Ca (mmol Sr:Ca ( Sr:Ca 200 20.00

0 0.00 0 500 1000 1500 2000 2500 3000 Distance (μm)

Figure 6. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #59.

26 0 1 2 3 4 5 6 1200 120.00 1050 100.00 900 80.00 750

600 60.00 mol mol-1) mol 450 40.00 300 Zn:Ca (mmol mol-1)Zn:Ca (mmol

Sr:Ca (μ Sr:Ca 20.00 150

0 0.00 0 500 1000 1500 2000 2500 3000 Distance (μm)

Figure 7. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #60.

27 0 1 2 3 4 5 6 7 1200 120.00

1050 100.00 900 80.00 750

600 60.00 mol mol-1) 450 40.00

300 mol-1) Zn:Ca (mmol

Sr:Ca (μ Sr:Ca 20.00 150

0 0.00 0 500 1000 1500 2000 2500 3000 3500 Distance (μm)

Figure 8. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #61.

28 7 6 5 4 3 2 1 0

1200 120.00 1050 100.00 900 80.00 750

600 60.00 mol mol-1) mol μ 450 40.00 300 Zn:Ca (mmol mol-1) Zn:Ca (mmol 20.00 Sr:Ca ( Sr:Ca 150

0 0.00 0 500 1000 1500 2000 2500 3000 Distance (μm)

Figure 9. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #62.

29 6 5 4 3 2 1 0 1 2 3 10000 160.00

9000 140.00 8000 120.00 7000 6000 100.00 5000 80.00 mol mol-1) mol μ 4000 60.00 3000 40.00 2000 mol-1) Zn:Ca (mmol Sr:Ca ( Sr:Ca 1000 20.00 0 0.00 0 500 1000 1500 2000 2500 3000 3500 Distance (μm)

Figure 10. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #63.

30 0 1 2 3 4 5 6 7 900 120.00 800 100.00 700 600 80.00 500 60.00

mol mol-1) mol 400 300 40.00

200 mol-1)Zn:Ca (mmol

Sr:Ca (μ Sr:Ca 20.00 100 0 0.00 0 500 1000 1500 2000 2500 3000 3500 Distance (μm)

Figure 11. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #64.

31 0 1 2 3 4 5 6 7

900 400.00

800 350.00

700 300.00 600 250.00 500 200.00

mol mol-1) mol 400 μ 150.00 300 100.00 200 mol-1) Zn:Ca (mmol Sr:Ca ( Sr:Ca 100 50.00 0 0.00 0 500 1000 1500 2000 2500 3000 3500 4000 Distance (μm)

Figure 12. Life history profile using Sr:Ca ratios (black) and Zn:Ca ratios (red) for bull trout #65.

32 1000 120.00 900 100.00 800 700 80.00 600 500 60.00 mol mol-1)

μ 400 40.00 300

Sr:Ca ( Sr:Ca 200 20.00 100 0 0.00 0 500 1000 1500 2000 2500 3000 Distance (μm)

Figure 13. Life history profile using Sr:Ca ratios (black) and Zn:Ca ratios (red) for bull trout #66.

33 0 1 2 3

16000 120.00

14000 100.00 12000 80.00 10000

8000 60.00 mol mol-1) mol 6000 40.00

4000 mol-1)Zn:Ca (mmol

Sr:Ca (μ Sr:Ca 20.00 2000

0 0.00 0 120 240 360 480 600 720 840 960 1080 1200 2640 2880 Distance (μm)

Figure 14. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #3.

34 0 1

1050 140.00

900 120.00

750 100.00

600 80.00

mol mol-1) 450 60.00 μ

300 40.00 Zn:Ca (mmol mol-1) (mmol Zn:Ca

Sr:Ca ( Sr:Ca 150 20.00

0 0.00 0 75 150 225 300 375 450 525 600 675 Distance (μm)

Figure 15. Life history profile using Sr:Ca ratios (yellow) and Zn:Ca ratios (red) for bull trout #26.

35 Appendix 2 Table 6. Estimated ages of all juvenile bull trout used in this study determined by Zn:Ca ratios. Sample Date Collected Source Life Stage Fork Length (mm) Zn Age 1 12-Sep-07 Halfway -26 km Juvenile 111 2+ 2 12-Sep-07 Halfway -26 km Juvenile 103 2+ 3 12-Sep-07 Halfway -26 km Juvenile 138 2+ 4 12-Sep-07 Halfway -26 km Juvenile 49 0+ 5 12-Sep-07 Halfway -26 km Juvenile 94 1+ 6 12-Sep-07 Halfway -26 km Juvenile 53 0+ 7 12-Sep-07 Halfway -26 km Juvenile 116 2+ 8 11-Sep-07 Halfway - HA5 Juvenile 99 2+ 9 11-Sep-07 Halfway - HA5 Juvenile 148 2+ 10 11-Sep-07 Halfway - HA5 Juvenile 110 2+ 11 11-Sep-07 Halfway - HA5 Juvenile 114 2+ 12 11-Sep-07 Halfway - HA5 Juvenile 168 3+ 13 11-Sep-07 Caribou Juvenile 54 0+ 14 11-Sep-07 Caribou Juvenile 54 0+ 15 11-Sep-07 Caribou Juvenile 62 0+ 16 11-Sep-07 Caribou Juvenile 63 0+ 17 11-Sep-07 Caribou Juvenile 62 0+ 18 11-Sep-07 Caribou Juvenile 68 0+ 19 11-Sep-07 Caribou Juvenile 69 0+ 20 11-Sep-07 Caribou Juvenile 66 0+ 21 11-Sep-07 Caribou Juvenile 61 0+ 22 11-Sep-07 Caribou Juvenile 61 0+ 23 11-Sep-07 Caribou Juvenile 144 2+ 24 11-Sep-07 McDonald Juvenile 115 2+ 25 11-Sep-07 McDonald Juvenile 183 3+ 26 11-Sep-07 McDonald Juvenile 56 0+ 27 11-Sep-07 McDonald Juvenile 115 1+ 28 11-Sep-07 McDonald Juvenile 104 1+ 29 11-Sep-07 McDonald Juvenile 113 1+ 30 11-Sep-07 McDonald Juvenile 61 0+ 31 11-Sep-07 McDonald Juvenile 115 1+ 32 11-Sep-07 McDonald Juvenile 149 2+ 33 11-Sep-07 McDonald Juvenile 182 3+ 34 10-Sep-07 Incomappleaux Juvenile 80 0+ 35 10-Sep-07 Incomappleaux Juvenile 62 0+ 36 10-Sep-07 Incomappleaux Juvenile 68 0+ 37 22-Oct-07 Incomappleaux Juvenile na 0+ 38 22-Oct-07 Incomappleaux Juvenile na 0+ 39 22-Oct-07 Incomappleaux Juvenile na 0+ 40 22-Oct-07 Incomappleaux Juvenile na 0+ 41 22-Oct-07 Incomappleaux Juvenile na 0+ 42 23-Oct-07 Kellie Ck Juvenile na 2+ 43 23-Oct-07 Kellie Ck Juvenile na 1+ 44 23-Oct-07 Kellie Ck Juvenile na 1+ 45 10-Sep-07 Illecillewaet Juvenile 118 1+ 46 10-Sep-07 Illecillewaet Juvenile 114 1+ 47 10-Sep-07 Illecillewaet Juvenile 58 0+ 48 10-Sep-07 Illecillewaet Juvenile 115 1+ 49 10-Sep-07 Illecillewaet Juvenile 99 1+ 50 10-Sep-07 Illecillewaet Juvenile 116 1+ 51 10-Sep-07 Illecillewaet Juvenile 104 1+ 52 10-Sep-07 Greeley Ck Juvenile 125 1+ 53 10-Sep-07 Greeley Ck Juvenile 124 1+ 54 10-Sep-07 Greeley Ck Juvenile 99 1+ 55 10-Sep-07 Greeley Ck Juvenile 104 ? 56 10-Sep-07 Greeley Ck Juvenile 96 1+