Acclimation Improves Short-Term Survival of Hatchery Lahontan Cutthroat Trout in Water From Saline, Alkaline Walker Lake, Nevada Free

Lahontan cutthroat trout Oncorhynchus clarkii henshawi, a federally listed “threatened” species (USOFR 1975), was extirpated from Walker Lake subsequent to the construction of Derby Dam in 1933, which blocked access of the lake population to important spawning habitat in the Walker River (La Rivers 1962; Sigler and Sigler 1987; Behnke 1992). Prior to extirpation, Lahontan cutthroat trout grew to sizes exceeding 18 kg and thrived in waters too saline (i.e., about 2,000 mg/L total dissolved solids [TDS]) for other trout species (Behnke 1992; Dickerson and Vinyard 1999). As a result of increased human use of ground and surface water in the Walker River Basin, the volume of Walker Lake decreased from 9 million acre-feet in 1882 to 4.5 million acre-feet in 2003 (USFWS 2003); TDS showed a corresponding increase from 2,500 mg/L to 14,600 mg/L (USFWS 2003).

For the past several decades, state and federal hatcheries have maintained a fishery in Walker Lake by annually stocking Lahontan cutthroat trout derived from out-of-basin strains. After 1995, hatchery Lahontan cutthroat trout were acclimated in water of intermediate salinity at Pyramid Lake, Nevada, prior to release in Walker Lake. After 2005, reduced relative inflows to Walker Lake compared to Pyramid Lake increased the disparity between their salinities to the point where acclimation at Pyramid Lake was considered ineffective. Bioassays in Walker Lake indicated short-term survival rates of Lahontan cutthroat trout acclimated in Pyramid lake decreased from >90% in 1995 to <5% in 2005 (NDOW 2005). The Lahontan National Fish Hatchery Complex produces Lahontan cutthroat trout for recovery efforts in Walker Lake, a saline and alkaline terminal lake in northwestern Nevada. The salinity and alkalinity of Walker Lake (>17,400 mg/L TDS and >9.5 pH, respectively) are so much higher than the well water utilized to produce trout at the Lahontan National Fish Hatchery in Gardnerville, Nevada (291–296 mg/L TDS and 7.5–7.8 pH, respectively), that new methods of acclimation are necessary. Dickerson and Vinyard (1999) found that challenges in Walker Lake water concentrated to 15,467 mg/L TDS quickly killed unacclimated subyearling Lahontan cutthroat trout derived from the contemporary Pyramid Lake stock and reared at Lahontan National Fish Hatchery. Acclimation in static water and increased fish size or age improved survival rates in unaltered lake water (then at 12,400 mg/L TDS and 9.4 pH; Dickerson and Vinyard 1999).

The U.S. Fish and Wildlife Service is evaluating the feasibility of constructing an acclimation facility on the shore of Walker Lake. The acclimation facility would provide a means of exposing hatchery Lahontan cutthroat trout to increasing concentrations of water from Walker Lake prior to their release. The simplest method of acclimation would be to transport fish and hatchery water from the hatchery to static lakeside tanks and repeatedly replace a portion of the hatchery water with lake water to increase the ratio of lake water to hatchery water. The most efficient rate, pattern, and duration of increasing the lake to hatchery water ratio need to be empirically determined. The objective of this study was to test the effect of fish size on survival, and the effectiveness of two acclimation protocols for 8-month-old Lahontan cutthroat trout reared at Lahontan National Fish Hatchery, in terms of reducing the hazard of mortality compared to that of unacclimated fish during a week-long challenge in water from saline, alkaline Walker Lake, Nevada.

Two thousand four hundred 8-month-old Lahontan cutthroat trout with a mean fork length (FL) of 143 mm (±17.6 mm SD) were randomly selected from the 2008 year class at Lahontan National Fish Hatchery, Gardnerville, Nevada. The fish were of the same age and size typically stocked in Walker Lake during February and March. All fish were fourth-generation progeny of trout captured from wild populations in the Pilot Mountains of western Utah confirmed by phylogenetic analysis as recent descendants from the extirpated Truckee River subbasin populations in Nevada and California (Peacock and Kirchoff 2007). All fish were reared in 12°C artesian well water under natural photoperiod. Fish were raised on Abernathy trout diet (Rangen Inc., Buhl, ID). Fish were taken off feed 2 d prior to transport and remained off feed throughout acclimation and the lake water challenge to reduce ammonia excretion rates in the static tanks. Fish were transported from the hatchery to a shoreline location (town of Walker Lake) in three 800-fish batches corresponding to 8- and 3-d acclimation treatments and unacclimated fish (0-d acclimation) on December 10, 15, and 18, 2008, respectively. Each batch of 800 fish was divided (with no known bias) into four 2,320-L tanks containing static hatchery water supplied with compressed oxygen. Tank walls and lids were insulated with 1.9-cm-thick R-Matte® Plus-3 (RMAX, Dallas, TX). Fish were allowed to adjust to the lakeside tanks in hatchery water for 24 h before any exposure to lake water, during which time there was no mortality.

Lake water was introduced into the 8- and 3-d acclimation treatment tanks beginning 24 h after transport on December 11 and 16, 2008, respectively. The ratio of lake to hatchery water was increased by pumping portions of water out of each tank and replacing it with lake water. For the 8-d treatment, 33% of the water was replaced with lake water on the first through fourth day of acclimation. For the 3-d acclimation treatment, 50% of the water was replaced with lake water on each day during acclimation. As a result, salinity and alkalinity increased at rates associated with acclimation treatment (Figure 1). All lake water was collected from the lake surface near the shore. No lake water was added to the 0-d acclimation tanks until the beginning of the challenge 24 h after transport. Temperatures in all tanks were monitored to the nearest 0.01°C at 30-min intervals with Stowaway Tidbit Temperature Loggers (Onset, Bourne, MA). Total ammonia was measured with a Nesslerization and colorimeter kit (LaMotte Co., Chestertown, MD) in each tank 24 h after the addition of fish. Dissolved oxygen concentrations were monitored with a Hach HQ10 oxygen meter (Hach Co., Loveland, CO) and maintained between 8 and 14 mg/L in all tanks. Electrical conductivity (EC) and pH were monitored once a day with a Hach sensION156 multiparameter meter. Electrical conductivity was standardized for temperature according to the linear relationship determined on site (EC  =  0.4177 × temperature + 11.776; N  =  79; r²  =  0.997). Electrical conductivity was converted to TDS as in Tracy (2004; TDS [mg/L]  =  0.8466 × EC [microsiemens/cm] at 25.0°C − 1,470.4). Mortalities were enumerated daily.

On December 19 at 1800 h, 76 fish were selected from each acclimation tank (with no known bias) to participate in a week-long lake water challenge. One extra tank was placed next to tank 1 in the row of 12 acclimation tanks. The extra tank was filled with 1,935 L of Walker Lake water and supplied with 76 fish netted from tank 1. Tank 1 was then drained, filled with 1,935 L of lake water, and supplied with 76 fish from tank 2. This process was repeated until fish were transferred from all of the acclimation tanks into lake water in adjacent tanks. The number of fish per tank and tank volume were chosen to match a preliminary study performed in static hatchery water, in which total ammonia (as nitrogen) concentrations in three tanks remained less than 0.2 mg/L for 1 wk. Fish not selected for the challenge were removed from the tanks and discarded. Temperature, pH, and EC measurements continued at the same frequency as during acclimation. Mortalities were removed and counted at 2-h intervals. Fork lengths of all fish were measured upon removal from the tanks.

The effects of the acclimation treatments and FL on the hazard of death were compared using a Cox proportional hazards model:

where h_i(t) was the hazard of death for the ith individual at time t; λ₀ was an unspecified baseline hazard function; β ^₃ and β ^₈ were the estimated effect parameters for 3- and 8-d acclimation treatments, respectively; β ^_FL was the effect parameter for fork length; x_i3 was equal to 1 if individual i was in the 3-d acclimation treatment and 0 otherwise; x_i8 was equal to 1 if individual i was in the 8-d acclimation treatment and 0 if otherwise; and x_iFL was the fork length of individual i. This model placed unacclimated fish in the reference group with both x_i3 and x_i8 equal to 0.

Raw data were arranged in a matrix with rows dedicated to individuals and a column designated for each variable (x_i3, x_i8, x_iFL, and time of death). Effect parameters were estimated by maximizing an equation based on the partial likelihood equation:

where t₁ < t₂ < … < t_D denoted the ordered times of death; β_k was the kth of p effect parameters, Z_(i)k was the kth of p covariates associated with the individual whose death was at time t_i; R(t_i) was the set of all individuals j who were still alive at time i − 1; and Z_jk was the kth of p covariates associated with individual j (Klein and Moeschberger 1997) but modified to accommodate ties in time of death according to the EXACT method as described by Allsion (1992).

The hazard ratio for each indicator variable was calculated as exp(β ^) and denoted the relative risk of death suffered by individuals for which that indicator variable was equal to 1 compared to individuals for which the indicator variable was equal to 0 when all other variables were of the same value. For example, if β ^₈  =  −1.2, then the hazard ratio (e^−1.2)  =  0.3, indicating fish in the 8-d acclimation treatment experienced 0.3 times the hazard of death suffered by unacclimated fish with the same fork length. For quantitative covariates (e.g., x_iFL) subtracting 1 from the hazard ratio yielded the estimated portion by which the hazard of death changes per unit increased in the covariate. The direction of change is indicated by the sign of the parameter estimate (Allison 1995). For example, if β ^_FL  =  −0.105, then the hazard ratio (e^−0.105) is 0.90 indicating each millimeter increase in fork length decreases the hazard of death by a portion of 0.10 (i.e., 0.90–1.00).

The Cox proportional hazards model was performed using SAS 9.2 PROC PHREG (SAS Institute, Cary, NC). Significance was set at α < 0.05. Pair-wise comparisons between tanks within treatments were performed using the PROC PHREG TEST function in SAS 9.2 as in Allison (1995). Tanks with dissimilar survival functions within treatments were omitted from the final model. One omitted tank (under the 0-d acclimation treatment) exhibited a higher mortality rate than the other control tanks apparently due to a water leak. The remaining omitted tanks (one under the 3-d acclimation treatment and two under the 8-d acclimation treatment) exhibited lower mortality rates than the other tanks in their respective treatments, apparently because snow inundation reduced their salinities after their covers were shifted by the wind. All omitted tanks would have favored the alternative hypothesis; therefore, their omission was conservative and slightly increased the hazard ratios and P-values associated with the 3- and 8-d acclimation treatments.

We investigated the effectiveness of two simple acclimation protocols for 8-month-old Lahontan cutthroat trout reared at Lahontan National Fish Hatchery in terms of reducing the hazard of mortality, compared to that of unacclimated fish, during a week-long challenge in water from saline, alkaline Walker Lake, Nevada, while controlling for differences in fork length. Water temperature decreased from 11.0°C to 2.5°C prior to the lake water challenge due to cold ambient temperatures. The temperature decrease was more rapid in the 0- and 3-d acclimation treatments than in the 8-d acclimation treatment (Figure 1). Total ammonia was less than 0.2 mg/L in all tanks 24 h after the addition of fish. Several fish in the 3-d acclimation treatment tanks exhibited aberrant behavior soon after the replacement of 50% of the hatchery water with lake water and the resulting increase in TDS (from <300 to >10,000 mg/L) and pH (from <7.6 to >9.5), including surface swimming with their heads out of water, loss of equilibrium, air-gulping, flared opercles, and sporadic bursts of rapid swimming resulting in collisions with the tank walls. These fish died during acclimation. Percent survival during acclimation (i.e., before the lake-water challenge) was not independent of treatment (mean ± SE: 0-d  =  98.9 ± 1.13%, 3-d  =  75.8 ± 11.3%, and 8-d  =  96.4 ± 0.45%; χ²  =  266.288; P < 0.001).

No fish survived the entire week-long challenge, during which mean salinity ranged from 17,459 to 17,819 mg/L TDS, and mean alkalinity ranged from 9.59 to 9.67 pH across treatments. Median challenge survival time for the 0-, 3-, and 8-d acclimation treatments was 8, 8, and 12 h, respectively. Maximum challenge survival time for the 0-, 3-, and 8-d acclimation treatments was 20, 36, and 110 h, respectively. Survival curves during the challenge, stratified by acclimation treatment, reflect a moderately beneficial effect of acclimation (Figure 2). Fish acclimated for 3 and 8 d experienced significantly reduced hazard of death compared to unacclimated fish (P < 0.0001; Table 1). Longer FL was associated with lower hazard of death during the challenge at an estimated rate of −0.027/mm increase in FL (P < 0.0001; Table 1). A few of the mortalities appeared to be precocious males; however, their incidence did not appear to be associated with treatment. Our results indicate the methods of acclimation used in this study will not produce acceptable survival rates for Lahontan cutthroat trout stocked in Walker Lake; however, the apparent beneficial effect of the 8-d acclimation treatment indicates the possibility of achieving higher survival rates pending improvements to the acclimation method.

Although all fish ultimately died before the end of the lake water challenge, our results indicate a moderately positive effect of acclimation and fish size on time to death of Lahontan cutthroat trout challenged with water from Walker Lake. The benefit of the 3-d acclimation treatment was offset by mortality during acclimation; however, the 8-d acclimation treatment appeared to impart an overall advantage. Dickerson and Vinyard (1999) also observed increased survival of Lahontan cutthroat trout challenged with Walker Lake water associated with acclimation and size when TDS concentrations were lower; however, unlike our results, 3-d acclimation produced higher survival rates than longer (6-d) acclimation. Our results suggest the acclimation method might be improved by the use of longer or older fish, longer acclimation, and better control of the water temperature, ammonia concentration, and alkalinity.

Tank water temperatures decreased at a faster rate during acclimation in the 3-d acclimation treatment than in the 8-d acclimation treatment (Figure 1). Rapidly decreasing temperature may have caused the higher mortality rates in the 3-d acclimation treatment or may have acted in conjunction with increasing TDS and pH. Temperature fluctuated between 5.0°C and 2.3°C during the challenge, but was similar across treatments. Surface temperatures in Walker Lake ranged from 7.6°C to 8.9°C during the challenge, and they do not commonly decrease to less than 6.0°C during February and March (Beutel et al. 2001) when hatchery fish releases occur. Temperature affects the ability of salmonids to acclimate to seawater (Saunders et al. 1975; Alexis et al. 1984; Virtanen and Oikari 1984; Staurnes et al. 2001) and may have decreased survival rates in our study. The proposed acclimation facility should employ more effective means of temperature control. Static tanks should be housed indoors. The use of flow through tanks for acclimation would probably provide sufficient temperature control in outdoor, insulated tanks.

Many fish (24.2%) in the 3-d acclimation treatment exhibited aberrant behavior indicative of ammonia toxicity (Post 1987) and mortality soon after initial exposure to 10,000 mg/L TDS, 9.68 pH, and rapidly decreasing temperature; however, fish in the 8-d acclimation treatment tolerated TDS >14,000 mg/L, pH >9.6, and gradually decreasing temperature for 6 d during acclimation without apparent difficulty. These results suggest the increase in TDS, pH, and/or the decrease in temperature was too rapid in the 3-d acclimation treatment. Future investigations should include comparisons of different rates and patterns of TDS and pH increases while acclimation duration and temperature are held constant, as well as comparisons of different acclimation durations, to help determine the most effective acclimation protocol.

Ammonia measurements taken during acclimation and the challenge were spoiled by sulfide interference and high pH; however, the higher mortality rate and behavioral symptoms exhibited by the 3-d acclimation treatment during acclimation suggest ammonia concentration in the water collected from Walker Lake for the 3-d acclimation treatment may have been higher than in the lake water collected for the 8-d treatment. Acute exposure to ammonia can result in ionoregulatory impairment, leading to reduced Na⁺ influx and K⁺ effusion in fish (Randall and Tsui 2002). The toxicity of total ammonia increases exponentially with alkalinity largely due to dissociation of ionized ammonia (NH₄⁺) to the un-ionized form (NH₃; Roberts 2001). Un-ionized ammonia can attack the central nervous system by crossing and modifying the blood–brain barrier and reducing cerebral adenosine triphosphate, resulting in hyperventilation, hyperexcitability, coma, convulsions, and death (Randall and Tsui 2002). Ammonia levels in the lake and tanks should be accurately monitored, using methods effective in seawater (e.g., USEPA 1997) throughout future studies to determine the relative contribution of fish excretions and lake water to ammonia concentrations. In static tanks, ammonia could be controlled by adding ChorAm-X® (Reed Mariculture Inc., Campbell, CA). The use of flow-through tanks would facilitate maintenance of challenge water that is more closely representative of lake ammonia concentrations and would improve the inference of challenge survival rates to rates achievable in the lake.

Surface ammonia concentrations in Walker Lake fluctuated spatially and temporally during the winter stocking seasons of 1993, 1995, and 1996, falling within the range of 0.000 to 0.250 mg/L (Beutel et al. 2001). Ammonia concentrations in Walker Lake are likely to increase with lake desiccation because the ratio of hypolimnetic volume to hypolimnetic oxygen demand decreases with reduced lake depth, resulting in longer periods of hypolimnetic anoxia during lake stratification (Beutel 2001). In laboratory exposures, the acute tolerance limit for total ammonia decreased from 181 mg/L at pH 6.51 to 2.53 mg/L at pH 9.01 in rainbow trout (O. mykiss; Thurston et al. 1981). Acute ammonia tolerances in westslope cutthroat trout at pH 8.0 were similar to rainbow trout (Thurston et al. 1978). We recommend the acute toxicity of ammonia be determined for Lahontan cutthroat trout in the saline, alkaline waters of Walker Lake so appropriate measures for ammonia control can be implemented during acclimation, and stocking can be avoided when lake ammonia concentrations are too high.

Our acclimation method produced a sudden, large increase in pH after the addition of only small amounts of lake water to the hatchery water because of the large buffering capacity of Walker Lake water (Figure 1). Lahontan cutthroat trout challenged with water from saline, alkaline Pyramid Lake appear to adapt quickly to increased pH by quickly increasing the number and size of chloride cells in their gills, where chloride anions may be exchanged for HCO₃⁻ (mitigated by ion uptake in the gills) to maintain lower internal pH than the alkaline waters (Wilkie et al. 1994). Acid–base regulation is important in maintaining safe internal ammonia concentrations. Lahontan cutthroat trout exposed to Pyramid Lake water exhibit chronic elevated plasma pH and ammonia concentrations, which maintain a partial pressure NH₃ gradient important for the passive branchial diffusion of ammonia from their blood to the water (Wright et al. 1993). A gradual increase in pH resulted in higher survival rates than sudden pH increases in hatchery-reared rainbow trout (Murray and Ziebell 1984). Our acclimation method may therefore be improved by creating a gradual increase in pH, allowing time for Lahontan cutthroat trout to increase the size and number of their branchial chloride cells. A gradual increase in pH could be accomplished in static tanks by initially adding minute amounts of lake water. This initial pH adjustment period could then be followed by a TDS acclimation period during which lake water could be added at a faster rate. A simultaneous, gradual increase of pH and TDS could be achieved in static water by using simulated lake water for acclimation. The use of simulated lake water would also allow acclimation to be performed at the hatchery without the risk of fish pathogen transfer from Walker Lake, facilitating temperature control without the need for a flow-through system. The effect of alkalinity should be examined separately from salinity to help determine if the increase of pH and TDS should be performed separately or simultaneously during acclimation.

The use of longer or older fish acclimated for an extended period of time should be investigated; however, there are trade-offs between the number of fish a hatchery can produce and the size and age to which the fish are grown. For such a program to be feasible, the increase in survival achieved by stocking longer or older fish would need to offset the decrease in numbers of fish stocked.

We conclude that acclimation appears to be necessary prior to the release of subyearling Lahontan cutthroat trout reared at Lahontan National Fish Hatchery into Walker Lake to achieve acceptable short-term survival rates. Improvements to our acclimation method should be investigated, including 1) better control of temperature and ammonia, 2) a more gradual increase in pH, 3) more effective rates and patterns of TDS and pH increases, and 4) the use of longer or older fish. Ammonia concentration should be monitored throughout acclimation, and the acute toxicity of ammonia to Lahontan cutthroat trout in Walker Lake water should be determined. Use of static tanks for acclimation offer the advantage of employment of simulated lake water to accomplish a simultaneous, gradual increase in TDS and pH, and the ability to acclimate at the hatchery where temperature can be controlled easily. Use of flow-through tanks at the lake side would allow more fish to be held in the tanks during acclimation and easier control of ammonia concentrations (assuming ammonia concentration in Walker Lake is not too high), and would facilitate inference of challenge results to the lake by eliminating effects of fish excretions and changes in water quality resulting from stagnation.

The salinity of Walker Lake (>17,400 mg/L) currently exceeds the salinity found by Dickerson and Vinyard (1999) to cause complete mortality in acclimated Lahontan cutthroat trout (i.e., 15,467 mg/L). They concluded: “Without adequate inflow of freshwater into the lake, we estimate that 100% mortality of Lahontan cutthroat trout stocked into Walker Lake within 48 h of stocking can be expected within 20 years” (p. 514). Our study presents no evidence with which to refute the conclusions of Dickerson and Vinyard (1999). Our results indicate stocking of Lahontan cutthroat trout into Walker Lake has become at best problematic and should not be continued until effective acclimation methods are developed or salinity decreases. While studies such as ours can address short-term survival rates, the determination of long-term survival rates through mark and recapture studies would provide a more complete assessment of the stocking program.

We thank Sarah Bigelow, Derek Bloomquist, Alvin Duncan, Scott Foott, James Hoang, Erik Horgen, Art and Barbara Jones, Ed Kelly, Tim Loux, Corene Luton, Roger Peka, Kris Urquhart, Cassidy Williams, and Karie Wright for their assistance with this study. We also wish to thank the Journal of Fish and Wildlife Management reviewers and Subject Editor for improving this article. This research was supported by the U.S. Fish and Wildlife Service Lahontan National Fish Hatchery Complex Fisheries Improvement Program.