Stress Indices in Migrating Juvenile Chinook Salmon and Steelhead of Wild and Hatchery Origin before and after Barge Transportation

Transactions of the American Fisheries Society 129:946 961, 2000 Copyright by the American Fisheries Society 2000 Stress Indices in Migrating Juvenile Chinook Salmon and Steelhead of Wild and Hatchery Origin before and after Barge Transportation JAMES L. CONGLETON* U.S. Geological Survey, Idaho Cooperative Fish and Wildlife Research Unit, Department of Fish and Wildlife Resources, University of Idaho, Moscow, Idaho, 83844-1141, USA WILLIAM J. LAVOIE Idaho Cooperative Fish and Wildlife Research Unit, Department of Fish and Wildlife Resources, University of Idaho, Moscow, Idaho, 83844-1141, USA CARL B. SCHRECK U.S. Geological Survey, Oregon Cooperative Fish and Wildlife Research Unit, Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331, USA LAWRENCE E. DAVIS Oregon Cooperative Fish and Wildlife Research Unit, Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331, USA Abstract. Migrating wild (W) and hatchery-reared (H) chinook salmon Oncorhynchus tshawytscha and steelhead Oncorhynchus mykiss juveniles were sampled after loading into fish-transport barges at Lower Granite Dam on the Snake River, Washington, and after barge transportation downstream to Bonneville Dam on the Columbia River. Stress indices (increased plasma cortisol and glucose concentrations and decreased plasma chloride concentrations) were higher (P 0.001) for chinook salmon sampled during midseason (early to mid-may), when fish loading densities in barges were at seasonal maximums, than were stress indices for those sampled earlier or later. Cortisol concentrations in chinook salmon were correlated with steelhead densities after loading of barges (P 0.0001, R 2 0.41) and after arrival of barges at Bonneville Dam (P 0.0001, R 2 0.65). Cortisol concentrations were not correlated with gill Na,K adenosine triphosphatase activities, which were higher in W than in H fish of both species. Cortisol concentrations were higher (P 0.0001 in 1994, P 0.02 in 1995) in W than in H chinook salmon, and concentrations declined in both groups during barge transportation early and late in the migration season but not during midseason. In contrast, cortisol concentrations were lower (P 0.001) in W than in H steelhead, were not correlated with steelhead loading densities, and declined in both W and H fish during barge transportation on all sampling dates. Electrolyte disturbances were greater in chinook salmon than in steelhead, but disturbances were similar for W and H fish of both species. Stressrelated water gain was, however, greater (or was compensated more slowly) in W than in H fish. These results indicate that chinook salmon are more stressed by barge transportation than are steelhead. If the viability of juvenile chinook salmon is reduced by adverse physiological, immunological, or behavioral responses to transportation stress, reductions in survival rates should be largest for fish transported during midseason, when densities of juvenile steelhead in the fishtransport barges are highest. The numbers of wild spring and summer chinook salmon Oncorhynchus tshawytscha and steelhead Oncorhynchus mykiss adults returning to the Snake River basin in Idaho and Oregon began to * Corresponding author: jconglet@uidaho.edu Received September 17, 1998; accepted January 27, 2000 decline in the mid-1970s and continued to decline through the mid-1990s. From 39,000 to 64,000 wild spring and summer chinook salmon returned annually during the years 1966 to 1973, but returning wild adults averaged only 5,400 from 1993 to 1998. Similarly, annual returns of wild steelhead ranged from 32,000 to 82,000 during the years 1964 to 1972 but averaged only 7,900 during the 946

CHINOOK SALMON STRESS INDICES 947 FIGURE 1. Locations of hydroelectric dams on the lower Snake River and lower Columbia River. Dams with facilities for collection and barge transportation of migrating juvenile salmonids are indicated by asterisks. years 1993 to 1998 (D. Cannamela, Idaho Department of Fish and Game, personal communication). As a result of these population declines, Snake River stocks of wild spring and summer chinook salmon (hereafter referred to as chinook salmon) and wild steelhead have been listed as threatened under the provisions of the Endangered Species Act. Factors believed to contribute to the decline of Snake River chinook salmon and steelhead runs include mortality of emigrating juvenile salmonids in the hydroelectric system on the Snake and Columbia rivers (Raymond 1979, 1988), predation on juveniles and adults in the estuary by growing populations of avian (C. B. Schreck and L. E. Davis, Oregon State University, personal communication; D. Roby, Oregon State University, personal communication) and mammalian (Harmon et al. 1994) predators, and decreased marine survival resulting from cyclic changes in weather and oceanic conditions (Mantua et al. 1997). To reduce exposure of migrating juvenile salmonids to the hydroelectric system, the U.S. Army Corps of Engineers collects between 11 and 21 million juveniles annually (Hetherman et al. 1998) at four dams on the Snake and Columbia rivers (Figure 1), transports them around the remaining downstream dams in trucks or barges, and releases them 235 km upstream from the mouth of the Columbia River. Transportation of juvenile fish eliminates mortalities caused by injury during powerhouse or dam passage, by exposure to water that is supersaturated with dissolved gases, and by predacious fish in the reservoirs. Although transported juveniles have returned at higher rates than juveniles migrating through the hydroelectric complex in the majority of trials (Ward et al. 1997), the transportation program (operational since the early 1980s) has not reversed the decline of chinook salmon and steelhead populations. Several agency and independent work groups have reviewed the efficacy of the transportation program. One often-mentioned hypothesis is that the survival of transported salmonids may be reduced as a result of the delayed effects of stressors experienced during collection and transportation. Exposure to stressors can disturb metabolic, hydromineral, and immune homeostasis and may adversely affect the fish s ability to cope with disease, predators, and other environmental challenges (Barton and Iwama 1991; Pickering 1993; Schreck et al. 1997; Wendelaar Bonga 1997). Published studies (Matthews et al. 1986; Maule et al. 1988) and unpublished agency reports have identified general and site-specific aspects of fish collection and transportation procedures that elicit stress responses. However, none of these studies has provided information on the stress responses of wild (W) fish, even though W fish are the focus of stock recovery efforts. Juvenile salmonids of W origin cannot be reliably distinguished from unmarked juveniles of hatchery (H) origin, and H juveniles comprise over 80% of emigrating chinook salmon and 90% of steelhead (Hetherman et al. 1998). For this reason, previous studies on mixed stocks of migrating W and H fish have pro-

948 CONGLETON ET AL. vided information that is useful for evaluating the stress responses of H fish, but not those of W fish, to collection and transportation. Comparison of the stress responses of migrating H and W fish is now possible, however, because in recent years juvenile chinook salmon and steelhead reared in hatcheries in the Snake River basin have been fin-clipped before release, thereby allowing for identification of H and W (unclipped) fish at downstream sampling sites. Several laboratory studies have compared concentrations of the stress hormone cortisol in juvenile chinook salmon and coho salmon Oncorhynchus kisutch from genetically similar W and H populations. In the studies of Mazur and Iwama (1993) and those of Shrimpton et al. (1994a, 1994b), W juveniles (chinook salmon and coho salmon, respectively) were migrating at the time of capture, but H juveniles were not, so that differences between the two groups in terms of mean plasma cortisol concentrations may have been related to differences in migratory behavior and stage of parr smolt development rather than to the rearing environment. Physiological changes characteristic of the parr smolt transformation are triggered or accelerated in migrating fish and include increases in gill Na,K adenosine triphosphatase (ATPase) activities (Zaugg et al. 1985; Rodgers et al. 1987), in resting plasma cortisol concentrations, and in the cortisol stress response to an imposed stressor (Specker and Schreck 1982; Barton et al. 1985; Young et al. 1989). Other studies have compared the cortisol stress response of W juvenile Pacific salmon held in captivity for periods ranging from 10 d to 7 months with the response of H fish (Mazur and Iwama 1993; Salonius and Iwama 1993) or have compared the stress responses of nonanadromous juvenile rainbow trout Oncorhynchus mykiss of W and H origin (Woodward and Strange 1987). In all comparisons of W and H fish, stress-induced increases in plasma cortisol and in secondary stress indices have been greater in W than in H fish. No comparisons have been made, however, between the stress responses or gill Na,K ATPase activities in actively emigrating W and H salmonids. The objectives of our study were to determine (1) if barge transportation is stressful for juvenile chinook salmon and steelhead; (2) if seasonal changes in the stage of parr smolt development affect stress responses to barge transportation; (3) if changes in fish loading densities affect stress responses to barge transportation; and (4) if stress responses differ for transported W and H fish. Chinook salmon were sampled during the 1992, 1993, 1994, and 1995 migrations, and steelhead were sampled during the 1994, 1995, and 1996 migrations. The endocrine stress response was evaluated by measurement of plasma cortisol, metabolic responses were evaluated by measurement of plasma glucose and liver glycogen, and ionoregulatory responses were evaluated by measurement of plasma chloride ion and sodium ion. The stage of parr smolt development was evaluated by measurement of gill Na,K ATPase activities. Methods Sampling procedures. Migrating fin-clipped (presumed H) and unclipped (presumed W) chinook salmon (n 12 of each) were sampled after they were loaded into the holds of fish-transport barges at Lower Granite Dam on six dates during the spring migration (mid-april to late May) in 1995. Samples were taken between 0800 and 1100 hours, immediately prior to departure of the barges. The fish had been loaded directly into the barges from the dam bypass system during the preceding 2 12 h, and they were expected to be in various stages of recovery following the loading process. Fish were again sampled from the same barge holds at Bonneville Dam after the barges had traveled 460 km downriver through six intervening dams (Figure 1). Barges arrived at Bonneville Dam 32 40 h after leaving Lower Granite Dam. Hatchery chinook salmon (n 20) were also sampled from gatewells at Lower Granite Dam and from barge holds at Lower Granite and Bonneville dams on three dates in each of the following years: 1992, 1993, and 1994 (sampling series 1994B). An additional series of samples was taken in 1994 to examine date-to-date variation in stress indices at shorter time intervals than had been employed in other years: fish were sampled from barge holds at Lower Granite Dam (but not at Bonneville Dam) on 10 dates (sampling series 1994A). Migrating H and W steelhead (n 16 of each) were sampled from barge holds at Lower Granite Dam and again at Bonneville Dam on six occasions in 1996; the procedures followed were identical to those for sampling of chinook salmon. In addition, H steelhead (n 20) were sampled from gatewells at Lower Granite Dam and from barge holds at Lower Granite and Bonneville dams on two occasions in 1994 and on three occasions in 1995. Fish were captured by lift net (Matthews et al. 1986) and immediately transferred to a 200 mg/l solution of buffered tricaine methanesulfonate, an anesthetic concentration that is quickly lethal and

CHINOOK SALMON STRESS INDICES 949 that is known to prevent postcapture changes in plasma cortisol (Barton et al. 1985). After recording any fin clips or freeze brands, fork length was measured to the nearest millimeter and weight to within 0.1 g. Condition factor was later calculated as weight (g) times 100 divided by length (cm) 3. Blood samples were taken from chinook salmon and from steelhead (in 1995) by severing the caudal peduncle and collecting blood (0.1 0.3 ml) in heparinized capillary tubes. In 1996, blood samples were taken from steelhead by puncturing the caudal vein with a 20-gauge needle and aspirating a sample (0.6 1.0 ml) into a heparinized syringe. Samples were centrifuged for 5 min at 1,200 gravity (g), and the plasma was immediately frozen on dry ice. Liver samples to be used for glycogen analysis (1995 chinook salmon only) were excised and frozen on dry ice. Gill filaments to be used for determination of Na,K ATPase activities were clipped from the first gill arch on the left side and frozen on dry ice in a buffer solution containing 0.3 M sucrose, 0.02 M disodium EDTA, and 0.1 M imidazole (ph 7.1). Blood and tissue samples were stored at 80 C prior to analysis. Analytical methods. Plasma cortisol concentrations were determined by a radioimmunoassay (Foster and Dunn 1974) modified for use with salmonid plasma (Redding et al. 1984). Cortisol concentrations were determined for plasma samples from individual fish in all years. Chloride ion concentrations were determined by use of an ion-specific electrode (920M chloride meter, Corning Laboratory Sciences, Corning, New York) in 1995 and by autoanalyzer (Dimension AR-1MT, DuPont, Wilmington, Delaware) in 1996. Chloride ion concentrations were determined for individual fish in 1994. Pooling was necessary in 1995 and 1996 to provide adequate sample volumes for determination of chloride ion, sodium ion, glucose, and other clinical indices not reported here. Subsamples (50 L for chinook salmon samples, 75 L for steelhead samples) were pooled in randomly selected sets of three to yield pooled sample volumes of 150 or 225 L. Sodium ion and glucose concentrations for pooled samples were determined by autoanalyzer (Dimension AR-1MT, DuPont). Liver glycogen concentrations were assayed using the method of Lo et al. (1970). Gill Na, K ATPase activities were assayed (courtesy of Dr. Alec Maule, U.S. Geological Survey) by the microassay modification (Schrock et al. 1994) of the deoxycholate extraction procedure described by Zaugg (1982), or they were assayed (Biotech Research and Consulting, Corvallis, Oregon) by the whole-homogenate procedure of Johnson et al. (1977). Specific activities estimated by the whole-homogenate procedure are considerably lower than specific activities estimated by the deoxycholate extraction procedure, but the estimates are proportional over a wide range of gill Na,K ATPase activities (R. Ewing, Biotech Research and Consulting, personal communication). Statistical analyses. We emphasized analysis of data for chinook salmon sampled in 1994 (series A) and 1995 and for steelhead sampled in 1996 for three reasons: both W and H fish were sampled in those years, sampling was performed more frequently in those years than in other years, and ionoregulatory and metabolic indices were measured in addition to plasma cortisol. Data were analyzed by two-way or three-way analysis of variance (ANOVA), with fish origin (H or W), sampling date, and (when appropriate) sampling location (Lower Granite Dam or Bonneville Dam) as fixed factors. If interactions were significant, data sets (i.e., H and W, or Lower Granite and Bonneville data sets) were analyzed separately. If the effect of sampling date was significant, means for individual dates were compared pairwise using Fisher s protected least significant difference (PLSD) test. Prior to ANOVA, data for steelhead sampled in 1996 (plasma cortisol, glucose, Na, and Cl ) were subjected to multivariate ANOVA (MANOVA). As a result of missing values, MAN- OVA could not be performed with data for chinook salmon that were sampled in 1994 or 1995. For this reason, the P level required for significance in ANOVA testing of 1994 and 1995 chinook salmon data was adjusted to 0.05 divided by k, where k was the number of dependent variables measured. As a result, required significance levels were P 0.025 in 1994 (two variables: cortisol and Cl ) and P 0.017 in 1995 (three variables: plasma cortisol, Cl, and glucose). The significance level was P 0.05 in all other statistical tests. Because some data sets for cortisol were heteroscedastic, they were analyzed both with and without log transformation; however, data transformation had little effect on estimates of statistical significance. Data sets for other physiological indices did not require transformation. As a result of relatively low plasma sample volumes for chinook salmon, samples were not available for some combinations of sampling date and sampling location, in which case three-way AN- OVAs could not be performed. In some instances, it was possible to analyze the H data set for a

950 CONGLETON ET AL. sampling date effect or to determine the significance of the factors of fish origin and sampling date at one sampling location but not at both. Models for the effects of selected independent variables on log-transformed cortisol concentrations in chinook salmon and steelhead were developed by stepwise backward multiple regression. The independent variables were year, sampling site (Lower Granite Dam or Bonneville Dam), fish origin (W or H), gill Na,K ATPase activity (because different assay procedures were used in different years, activities were scaled proportionally to peak activities in the May 17 23 period), loading density of chinook salmon, and loading density of steelhead trout. Cortisol data for all years were used in these analyses. Results Migration Timing and River Conditions Two major peaks in numbers of juvenile steelhead migrants occurred in 1994, 1995, and 1996, the first in late April or early May and the second in mid-may (Figure 2). Two peaks in the numbers of chinook salmon migrants (not obvious in 1996, when numbers were very low) occurred concurrently with the peaks in steelhead numbers. In addition, an earlier peak in chinook salmon migrants occurred 5 7 d in advance of the first steelhead peak. Snake River flows during the spring out-migration period ( 90% of migrating fish passing from April 20 to May 20) increased from year to year from 1994 to 1996 (Figure 2). Snake River temperatures at Lower Granite Dam were warmer in the low-flow year of 1994 (11 15 C) than in 1995 or 1996 (9 13 C in 1995; 6 10 C in 1996). Columbia River temperatures at Bonneville Dam ranged from 8 to 13 C in mid-april, rising to 17 C by mid-may in 1994 and to 12 15 C by mid-may in 1995 and 1996. FIGURE 2. Numbers of juvenile steelhead and spring/ summer chinook salmon entering the fish collection facility each day and mean daily river discharge (KCFS; 1,000 ft 3 /s) at Lower Granite Dam during the period from April 20 to May 30 in the years 1994, 1995, and 1996 (Fish Passage Center, Portland, Oregon). Lengths, Weights, and Condition Factors of W and H Fish Juvenile chinook salmon and steelhead of H origin were longer and heavier (P 0.0001) than their W counterparts (Table 1). Condition factors did not differ for W and H chinook salmon in 1994, but they were higher (P 0.0001) for W than for H fish in 1995 (Table 1). The condition factors of H chinook salmon declined progressively during the migration seasons in 1994 and 1995 (P 0.01 for date effect in both years), but condition factors of W fish did not decline during the migration season in either year. Condition factors were higher for W than for H steelhead (P 0.001) in 1996; as with chinook salmon, condition factors of H fish, but not those of W fish, declined (P 0.0001) over time. Gill Na,K ATPase Activities Intraseasonal trends. Gill Na, K ATPase activities in H chinook salmon were lowest in the earliest samples taken (April 23 and April 21) in 1994 and 1995, increasing thereafter (P 0.001 for date effect in both years) until early May, with little change in the following weeks (Figure 3). Gill Na,K ATPase activities in W fish did not vary over time in either year. Gill Na,K ATPase activities in both H and W steelhead (fish origin date interaction, P 0.65) were lowest in the earliest samples taken (May 1 and 7) in 1996, increasing thereafter (P 0.004), with little change after mid-may (Figure 3).

CHINOOK SALMON STRESS INDICES 951 TABLE 1. Mean ( SE) fork lengths (mm), weights (g), and condition factors for hatchery and wild chinook salmon smolts sampled in 1994 and 1995 and for hatchery and wild steelhead smolts sampled in 1996. Parenthetical values are sample sizes. Species Chinook salmon Steelhead Chinook salmon Steelhead Chinook salmon Steelhead Sample year Hatchery Wild 1994 1995 1996 1994 1995 1996 1994 1995 1996 Fork length (mm) 138 1.0 (127) 135 1.0 (148) 217 1.5 (161) Weight (g) 25.8 0.6 (126) 24.0 0.6 (148) 91.9 1.9 (161) Condition factor 0.96 0.006 (126) 0.94 0.005 (148) 0.88 0.004 (161) 116 1.2 (110) 109 0.8 (143) 176 2.0 (148) 15.4 0.5 (109) 13.0 0.3 (143) 51.2 1.8 (148) 0.96 0.005 (109) 0.98 0.006 (143) 0.90 0.006 (148) Comparison of H and W fish. In 1994, gill Na, K ATPase activities were lower (P 0.0001) for H than for W chinook salmon (Figure 3). In 1995, gill Na,K ATPase activities were lower (P 0.01) for H than for W chinook salmon in samples taken on and before May 3 4, 1 but activities were similar in samples taken on and after May 9 10. Gill Na,K ATPase activities were lower (P 0.0001) for H than for W steelhead throughout the out-migration season in 1996 (Figure 3). Stress Indices Intraseasonal trends. Plasma cortisol concentrations in both H and W chinook salmon were relatively low ( 75 ng/ml) in the earliest samples taken in 1994 and 1995 (Figures 4, 5), increasing thereafter (P 0.001 for date effect in both years and, in 1995, at both sampling locations) to reach maximum levels (ranges for three highest mean concentrations of 226 239 ng/ml for W fish and 150 172 ng/ml for H fish) in late April and early May, then declining significantly by the third week of May. In 1994, two seasonal cortisol peaks were evident, with significantly higher cortisol concentrations on April 29, May 2, and May 11 than on other sampling dates. In 1995, cortisol concentrations were significantly higher on May 3 4 and May 9 10 than on other sampling dates in W fish at both dams and in H fish at Bonneville Dam. Additional data for H chinook salmon from the years 1992, 1993, and 1994 indicated intraseasonal changes similar to those shown by the more de- 1 Hyphenated dates are those on which samples were taken from barges at Lower Granite Dam and again (the following day) at Bonneville Dam. tailed 1994 and 1995 sampling series: cortisol concentrations were higher (ANOVA, P 0.03) at both sampling locations during midseason (May 5 17; n 10 samples for combined locations; overall mean SE 148 17 ng/ml) than they were earlier (April 22 28; n 6 samples; overall mean 74 9.5 ng/ml) or later (May 24 25; n 2 samples; overall mean 70 29 ng/ml). Mean cortisol concentrations for H chinook salmon sampled from gatewells on nine dates in 1992, 1993, and 1994 ranged from 53 to 160 ng/ml (overall mean 74 12 ng/ml, median 67 ng/ml), and concentrations did not differ significantly among early-, middle-, and late-season samples. Plasma Cl concentrations in both H and W chinook salmon were relatively high (131 and 132 meq/l) in the earliest samples taken in 1994 and 1995 (Figures 4, 5), decreasing (P 0.002 for date effects) to minimum levels (99 106 meq/l) in late April and early May, and then increasing significantly by the third week in May. In both years, seasonal low Cl concentrations occurred on those dates when plasma cortisol concentrations were at or near seasonal highs. Plasma glucose concentrations differed (P 0.006) between dates in 1995 (Figure 5) and were highest (166 273 mg/dl in H chinook salmon, 108 141 mg/dl in W fish) on May 3 4 and May 9 10, dates during which plasma cortisol concentrations were also elevated. Mean glucose concentrations ranged from 66 to 99 mg/dl on other dates. For steelhead, a MANOVA with cortisol, Na, Cl, and glucose data indicated that the effects of all factors fish origin, sampling site, and sam-

952 CONGLETON ET AL. FIGURE 4. Mean ( SE) plasma cortisol and chloride ion concentrations for hatchery (H; striped bars) and wild (W; solid bars) chinook salmon sampled from fishtransport barges at Lower Granite Dam (LGr) in 1994. Cortisol concentrations were higher (P 0.0001) in W than in H fish. Chloride ion concentrations did not differ in H and W fish: composite data are shown. Date effects indicated as described in Figure 3. FIGURE 3. Mean ( SE) gill Na,K ATPase activities for hatchery (H; striped bars) and wild (W; solid bars) chinook salmon sampled from fish-transport barges at Lower Granite Dam in 1994 and at Lower Granite and Bonneville dams in 1995 (data for the two sites did not differ: composite data are shown) and for H and W steelhead sampled from barges at Lower Granite Dam in 1996. The deoxychlolate extraction method was used in 1994, and the whole-homogenate preparation method (which yields lower specific activities) was used in 1995 and 1996. Activities were lower (P 0.0001 overall in 1994; P 0.01 for the period from April 21 to May 4 in 1995) in H than in W chinook salmon, and activities were lower (P 0.0001) in H than in W steelhead. Means for different dates within each series (H or W) were compared using Fisher s PLSD test; those not differing significantly are identified by superscripted letters. If date effects did not differ for H and W fish, one set of underlined letters is shown. The absence of letters indicates the absence of a significant date effect. pling date were highly significant (P 0.001; Wilk s lambda 0.73, 0.30, and 0.60), as was the sampling site by sampling date interaction. Separate ANOVAs were performed for each of the stress indices. The ANOVA with steelhead cortisol data indicated a highly significant interaction between sampling date and sampling location; therefore, data for the two locations were analyzed separately. Cortisol concentrations in both H and W steelhead sampled at Lower Granite Dam were relatively low (46 87 ng/ml) during the first week of May, and they trended upward (P 0.0001) to higher levels (ranges for three highest mean concentrations, 77 104 ng/ml for W fish, 110 118 ng/ml for H fish) by mid- to late May (Figure 6). An intraseasonal trend was not evident in steelhead sampled at Bonneville Dam. Additional cortisol data for H fish sampled from barges and from gatewells in 1994 and 1995 (total of five samples at each sampling location) were not adequate for evaluation of intraseasonal trends. Mean cortisol concentrations

CHINOOK SALMON STRESS INDICES 953 FIGURE 5. Mean ( SE) plasma cortisol, chloride ion, and glucose concentrations for hatchery (H; striped bars) and wild (W; solid bars) chinook salmon sampled from fish-transport barges at Lower Granite Dam (LGr) and Bonneville Dam (Bonn) in 1995. Cortisol concentrations were higher (P 0.0001) in W than in H fish, and they were higher (P 0.01) at LGr than at Bonn. Chloride ion concentrations did not differ for H and W fish or between sampling sites, and glucose concentrations did not differ between sampling sites: composite data are shown for these indices. Date effects indicated as described in Figure 3. FIGURE 6. Mean ( SE) plasma cortisol, sodium ion, and chloride ion concentrations for hatchery (H; striped bars) and wild (W; solid bars) steelhead sampled from fish-transport barges at Lower Granite Dam (LGr) and Bonneville Dam (Bonn) in 1996. Cortisol concentrations were higher (P 0.001) in H than in W fish at both locations and were higher (P 0.0001) at LGr than at Bonn. Sodium ion and chloride ion concentrations did not differ for H and W fish or between sampling sites: composite data are shown for these indices. Date effects (which were identical for sodium and chloride ion concentrations) indicated as described in Figure 3. for H steelhead sampled from gatewells ranged from 17 to 75 ng/ml (overall mean SE 34 12 ng/ml, median 17 ng/ml). Plasma Na and Cl concentrations in both H and W steelhead were relatively high (169 173 meq/l for Na, 138 142 meq/l for Cl ) at both sampling sites during the first week in May 1996, decreasing (P 0.01 for both ions) to lower levels (160 165 meq/l for Na, 131 134 meq/l for Cl ) on May 13 14 and May 19 20 (Figure 6).

954 CONGLETON ET AL. The lowest Na and Cl concentrations occurred on those dates when plasma cortisol concentrations in fish sampled at Lower Granite Dam were at or near seasonal highs. The ANOVA for plasma glucose concentrations in steelhead indicated a significant interaction between sampling date and location. Separate analyses for the two locations indicated that glucose concentrations in both H and W steelhead sampled at Lower Granite Dam were relatively low (78 mg/ dl for W fish, 116 mg/dl for H fish) on May 1, increasing (P 0.006) to higher levels (111 157 mg/dl for W fish, 166 177 mg/dl for H fish) on May 13 and May 19, thus coinciding with dates on which cortisol concentrations were relatively high and on which Na and Cl concentrations were relatively low. Plasma glucose concentrations decreased significantly, however, by May 25 (117 mg/dl for W fish, 124 mg/dl for H fish), although cortisol concentrations remained relatively high. An intraseasonal trend in glucose concentrations was not evident in steelhead sampled at Bonneville Dam: seasonal averages were 82 mg/ dl for W fish and 117 mg/dl for H fish. Comparison of H and W fish. In 1994, cortisol concentrations were higher (P 0.0001) in W than in H chinook salmon throughout the season (Figure 4). In 1995, cortisol concentrations were again higher (P 0.02) in W than in H chinook salmon overall, but differences were apparent only in midseason samples (Figure 5), resulting in a significant interaction between the factors of fish origin and sampling date. Plasma Cl concentrations were similar in H and W fish throughout the 1994 out-migration (Figure 4); glucose concentrations were not determined. In 1995, too few Cl and glucose analyses were completed from W fish sampled at Lower Granite Dam to allow for comparison with H fish, but Cl and glucose concentrations did not differ for H and W fish sampled at Bonneville Dam. Differences in plasma glucose concentrations for H and W fish were, however, nearly significant (P 0.05; P 0.017 required for significance because of multiple dependent variables) on May 3 4 and on May 9 10, with concentrations that were 58 and 132 mg/dl higher in H fish (Figure 5). These dates correspond to dates on which other stress indices were elevated in both W and H chinook salmon. Liver glycogen concentrations in H and W chinook salmon did not differ in 1995, averaging 1.38 ( 0.12 SE) mg/g in H fish and 1.32 ( 0.08 SE) mg/g in W fish. Cortisol (Figure 6) and glucose concentrations were consistently higher (P 0.001) in H than in W steelhead throughout the season in 1996. Plasma Na and Cl concentrations did not differ between H and W steelhead sampled at either Lower Granite or Bonneville dams. Changes in stress indices during barge transportation. Overall, plasma cortisol concentrations were lower (P 0.004) in W and H chinook salmon sampled at Bonneville Dam than in those sampled at Lower Granite Dam in 1995 (Figure 5). However, cortisol concentrations remained almost unchanged during barge transportation on May 3 4 and on May 9 10, resulting in a significant sampling date by sampling location interaction. Other stress indices (plasma Cl and glucose, liver glycogen) did not differ between sampling locations. Mean cortisol concentrations did not differ significantly between the two sites for H chinook salmon sampled in 1992, 1993, and 1994 (n 9 samples at each site; mean SE 134 15 ng/ml at Lower Granite Dam, 99 22 ng/ml at Bonneville Dam; P 0.14). Plasma cortisol concentrations were lower (P 0.0001) in steelhead sampled at Bonneville Dam than in those sampled at Lower Granite Dam on all sampling dates in 1996 (Figure 6). Plasma glucose concentrations also declined significantly (P 0.0001) in H and W fish during barge transportation, averaging 131 ( 6.3 SE) mg/dl at Lower Granite Dam and 101 ( 5.5 SE) mg/dl at Bonneville Dam. Plasma Na and Cl concentrations did not differ between sampling locations. Additional data for H steelhead sampled in 1994 (two samples at each site) and 1995 (three samples at each site) indicated that cortisol concentrations declined during barge transportation on all sampling dates (paired t-test, P 0.05; mean: 82 ng/ml at Lower Granite Dam, 46 ng/ml at Bonneville Dam). Condition factors of W chinook salmon were significantly lower (P 0.05) for fish sampled at Bonneville Dam than for those sampled at Lower Granite Dam in 1995. The largest differences between the sites occurred on May 3 4 and May 9 10 (Figure 7), dates during which stress responses were at seasonal extremes (Figure 5). Similarly, condition factors of W steelhead were significantly lower (P 0.001) for fish sampled at Bonneville Dam than for those sampled at Lower Granite Dam in 1996. The largest differences occurred on May 13 14 and May 19 20 (Figure 7), dates on which stress responses were relatively elevated (Figure 6). Condition factors of H chinook salmon and H steelhead did not differ between the two sampling locations.

CHINOOK SALMON STRESS INDICES 955 TABLE 2. Barge loading densities by species for dates when chinook salmon (CS) or steelhead (SH) were sampled from barge holds at Lower Granite Dam and after transportation to Bonneville Dam. FIGURE 7. Differences in mean condition factors for juvenile salmonids sampled before and after barge transportation (i.e., mean at Lower Granite Dam minus mean for same group at Bonneville Dam) on various dates in 1995 (chinook salmon) and 1996 (steelhead). Data for hatchery fish are indicated by striped bars, and data for wild fish are indicated by solid bars. Date 1992 (CS) Apr 27 May 5 May 17 1993 (CS) Apr 23 May 5 May 17 1994B (CS, SH) Apr 25 May 7 May 23 1994A (CS) Apr 23 Apr 26 Apr 29 May 2 May 5 May 8 May 11 May 14 May 17 May 20 1995 (CS) Apr 21 Apr 27 May 4 May 9 May 13 May 19 1995 (SH) May 6 May 17 May 31 1996 (SH) May 1 May 7 May 13 May 19 May 25 Barge loading density (g/l) Chinook salmon 5.7 2.4 1.0 2.2 6.3 3.8 12.6 6.4 0.4 2.5 7.2 3.0 4.2 2.1 4.8 4.1 1.4 1.9 1.6 10.4 3.5 5.5 5.2 3.7 1.8 9.8 1.9 1.2 0.2 0.7 2.1 0.9 0.3 Steelhead 9.4 53.6 23.5 9.9 50.8 54.0 34.4 46.9 8.8 12.3 42.8 43.4 43.8 21.9 53.2 53.3 25.4 22.3 25.1 10.5 19.4 60.8 49.2 29.3 14.7 51.0 13.0 11.3 5.4 24.0 25.0 18.1 8.0 Stress indices and fish densities in barges. Models for the effects of selected independent variables on log-transformed cortisol concentrations in chinook salmon sampled from barges were developed by stepwise backward multiple regression. The independent variables were sampling site (Lower Granite Dam or Bonneville Dam), fish origin (W or H), year (1992, 1993, 1994, or 1995), gill Na, K ATPase activities (scaled proportionally to peak activities in the May 17 23 period for each year), loading density of chinook salmon in barge holds (range 0.4 13 g/l; Table 2), and loading density of steelhead trout (range 9 61 g/ L; Table 2). Three variables steelhead density, chinook density, and sampling site accounted for 47% of the variability in chinook salmon cortisol concentrations (R 2 0.47, P 0.0001; Table 3). When data for the two sites were analyzed separately (Table 3), only steelhead density was retained as an independent variable in the model for cortisol concentrations in chinook salmon arriving at Bonneville Dam (R 2 0.65, P 0.0001). Stepwise backward multiple regression analysis to identify independent variables affecting plasma cortisol concentrations in steelhead (the same variables used in the analysis for chinook salmon were initially entered) did not indicate any significant relationships. Discussion Juvenile chinook salmon were stressed by cotransportation in barges with juvenile steelhead

956 CONGLETON ET AL. TABLE 3. Multiple-regression models for effects of selected variables on cortisol concentrations (log transformed) in juvenile chinook salmon (CS) cotransported with juvenile steelhead (SH) and sampled from barges at Lower Granite Dam and at Bonneville Dam, 1992 1995. Site and variable Both sites a Intercept SH density CS density Sampling site (Regression) Lower Granite Dam b Intercept SH density CS density (Regression) Bonneville Dam c Intercept SH density a df 3, 59. b df 2, 40. c df 1, 18. Coefficient 1.83 0.009 0.021 0.124 1.93 0.008 0.032 1.52 0.012 Standard error P R 2 0.057 0.001 0.009 0.050 0.065 0.002 0.010 0.081 0.002 0.0001 0.0001 0.02 0.02 0.0001 0.47 0.0001 0.0001 0.003 0.0001 0.41 0.0001 0.0001 0.65 when steelhead densities were high. Plasma cortisol concentrations declined in chinook salmon transported by barge early (late April) and late (after mid-may) in the 1992, 1993, 1994, and 1995 migration seasons, but concentrations remained elevated or increased in chinook salmon transported in midseason. During the midseason period, fish densities in the barges were at seasonal maximums (55 65 g/l), with over 90% of the fish weight per unit volume contributed by juvenile steelhead. The possibility of behavioral interactions between chinook salmon and steelhead during collection and transportation has been recognized for some time (Matthews et al. 1986), and behavioral and physiological evidence indicates that chinook salmon confined with steelhead are stressed by aggressive interactions initiated by the larger and more aggressive steelhead (Kelsey 1997). Multiple regression analyses indicated that cortisol concentrations in chinook salmon sampled from barges at both Lower Granite and Bonneville dams were positively correlated (P 0.0001 at both sites) with densities of steelhead. In addition to the positive correlation between cortisol concentrations in chinook salmon and steelhead densities, cortisol concentrations in chinook salmon were negatively correlated (P 0.003) with chinook salmon densities. This effect was evident in chinook salmon sampled at Lower Granite Dam, but not in those sampled at Bonneville Dam. An explanation may be provided by the observation of Kelsey (1997), who found that chinook salmon held in laboratory tanks schooled more tightly in the presence of steelhead. If this behavior also occurs in barged fish, it would be facilitated by higher densities of chinook salmon, and it could reduce the frequency of interaction between individuals of the two species. Kelsey (1997) also found that steelhead began to occupy and aggressively defend territories several hours after introduction into new surroundings. Aggressive behavior by steelhead may have increased during barge transportation, thereby strengthening the positive correlation between cortisol concentrations in chinook salmon and steelhead density (R 2 0.41 at Lower Granite Dam, R 2 0.65 at Bonneville Dam) and overriding the negative correlation with chinook salmon density. All stress indices plasma cortisol, glucose, and chloride ion varied significantly over time for chinook salmon sampled from barges at Lower Granite Dam (prior to barge transportation). Intraseasonal peaks in stress indices occurred during the first half of May; these peaks corresponded with the passage of peak numbers of juvenile steelhead through the Lower Granite facility and maximum loading densities of steelhead in barges. Elevated stress indices at these times may reflect exposure of chinook salmon to steelhead at various points in the collection system prior to barge loading as well as during the several hours that individuals of the two species were held together in barge holds before sampling. Juvenile salmonids are exposed to a variety of hydraulic, crowding, and air-exposure stressors during the collection and loading process (Matthews et al. 1986; Maule et al. 1988); however, the only potential stressor known to increase in prominence during the middle period of the migration season and to decrease thereafter is steelhead density. Intraseasonal variations in cortisol concentrations were not significantly correlated with changes in gill Na,K ATPase activities in either chinook salmon or steelhead. Although resting and poststress cortisol concentrations are higher in salmonid smolts than in parr (Barton et al. 1985; Young et al. 1989), all the fish sampled at Lower Granite Dam had begun the downstream migration several weeks earlier, and so were well advanced in the parr smolt transformation. Between-date differences in smoltification status may not have been large enough to significantly affect plasma cortisol concentrations. Juvenile steelhead did not appear to be stressed by barge transportation. Plasma cortisol concentrations declined to relatively low concentrations

CHINOOK SALMON STRESS INDICES 957 ( 50 ng/ml on 13 of 15 occasions) in both W and H steelhead transported by barge in 1994, 1995, and 1996, indicating recovery from earlier exposure to stressors during collection and barge loading. Cortisol and glucose concentrations increased and electrolyte concentrations decreased significantly after the first week of May, but the changes were relatively small and were not correlated with fish loading densities or any of the other factors examined. Fin-clipped chinook salmon and steelhead sampled during this study were known with certainty to be H fish, but some unclipped (nominal W) fish could have been H fish with regenerated fins or H fish that had never been fin-clipped. A few unclipped chinook salmon (six in 1994 and two in 1995) were carrying coded wire tags that identified them as H fish; these fish were reassigned to the H category. The number of additional fish incorrectly classified as W is not known. Fin clips were not recognizable in 4% of recently clipped juvenile chinook salmon examined at Dworshak National Fish Hatchery (Ahsahka, Idaho) in 1995 and in 0.2 1.1% of adult steelhead returning to the hatchery in the years 1992 through 1999 (R. Roseberg, U.S. Fish and Wildlife Service, personal communication). Because H fish make up 80 90% of emigrating chinook salmon and steelhead, the population of nominal W fish would include approximately 3 7% H fish if 1% of H fish had unrecognizable clips, and it would include 12 28% H fish if 4% of H fish had unrecognizable clips. Estimates of physiological indices for W fish reported here are likely to be somewhat biased by inclusion of H fish in the W category; the result would be to underestimate physiological differences between W and H fish. Cortisol concentrations differed significantly between W and H chinook salmon and between W and H steelhead. Peak cortisol concentrations (ranges for three highest daily means) for fish sampled from barges were 226 239 ng/ml for W chinook salmon, 150 172 ng/ml for H chinook salmon, 110 118 ng/ml for H steelhead, and 77 104 ng/ml for W steelhead. Cortisol response ranges (peak values minus baseline values for unstressed fish) for W and H fish could not be determined, because it was not possible to obtain samples of unstressed fish prior to collection at Lower Granite Dam. No published data are available on baseline cortisol concentrations in emigrating H salmonids, and few data are available for emigrating W salmonids. Peak springtime cortisol concentrations reported for nonmigrating, yearling H coho salmon smolts have ranged from approximately 40 to 70 ng/ml (e.g., Barton et al. 1985; Shrimpton et al. 1994b). These reported concentrations are similar to concentrations measured in migrating H chinook salmon collected from gatewells at Lower Granite Dam in the present study (median 67 mg/ml), but they are higher than cortisol concentrations measured in H steelhead collected from gatewells (median 17 ng/ml). Cortisol concentrations in migrating W chinook salmon (Mazur and Iwama 1993; Salonius and Iwama 1993) and coho salmon (Shrimpton et al. 1994a, 1994b) captured at smolt enumeration fences have ranged from approximately 60 to 120 ng/ml. However, when migrating W chinook salmon smolts sampled in the field were held in laboratory tanks for 10 d prior to a second sampling, cortisol concentrations declined from approximately 120 to approximately 20 ng/ml (Mazur and Iwama 1993). Thus, it is not certain that migrating smolts that are captured after they have encountered barriers to downstream migration are unstressed. The use of special sampling methods may be necessary to obtain reliable measurements of baseline cortisol concentrations in migrating juvenile salmonids. Stress responses in different stocks or species of fish should not be compared only on the basis of cortisol concentrations (Barton and Iwama 1991); the severity of disturbances in metabolic, osmoregulatory, and various other measures of performance may better reflect possible adverse effects. In the present study, plasma glucose, Na, and Cl concentrations were measured in addition to plasma cortisol. Glycogenolytic production of glucose may have been limited, for both H and W chinook salmon, by the availability of substrate: liver glycogen concentrations were low on all dates, varying from 1.0 to 1.75 mg/g wet liver weight (liver glycogen was not determined for steelhead). The higher peak glucose levels in H fish may have been a consequence of greater stressinduced gluconeogenic catabolism of body lipid stores: coelomic fat reserves were consistently present in H chinook salmon and steelhead, but they were absent in W fish in all years of the study. Ionoregulatory disturbances were small in both W and H steelhead following collection and barge transportation: the highest observed mean plasma electrolyte concentrations of 169 173 meq/l for Na and 138 142 meq/l for Cl were near the upper end of the range of values reported for undisturbed juvenile salmonids in freshwater (Folmar and Dickhoff 1981), and these values did not differ greatly from the lowest mean values of 160

958 CONGLETON ET AL. 165 meq/l for Na and 131 134 meq/l for Cl. In contrast, intraseasonal ranges for plasma Cl (Na was not measured) were much greater for chinook salmon: the highest mean concentrations were 131 132 meq/ml, and the lowest ranged from 99 to 106 meq/ml. Electrolyte loss is believed to be a proximate cause of death in severely stressed salmonids (McDonald and Milligan 1997). Mortality rates observed at the Lower Granite Dam fish collection facility are higher for juvenile chinook salmon than for steelhead (average mortalities for 1993 through 1997 were 0.5% of chinook salmon collected versus 0.1% of steelhead; Hetherman et al. 1997); this difference may be a consequence of larger ionoregulatory disturbances in stressed chinook salmon. Plasma Na and Cl concentrations did not differ between W and H chinook salmon or between W and H steelhead either before or after barge transportation. In the critical area of ionoregulatory performance, W and H fish performed similarly. Mortality rates for W and H chinook salmon and for W and H steelhead at the Lower Granite Dam fish facility were also quite similar (Hetherman et al. 1997). Gill Na,K ATPase activities for migrating H steelhead and chinook salmon were significantly lower than for their W counterparts. Differences between H and W fish were greatest in late April to early May, but they were evident as late as the third or fourth week of May. Suppression of the springtime rise in gill Na,K ATPase activity has been reported for H salmonids reared in raceways at higher densities (Patino et al. 1986), but the observation that gill Na,K ATPase activities were lower in H salmonids than in W fish 5 7 weeks after release of the H fish suggests that the effects of hatchery rearing on smolt development can be long-lasting. Gill Na,K ATPase is directly involved in ionic extrusion in seawater (Silva et al. 1977) and has been shown in some studies to be correlated with seawater tolerance (McCormick et al. 1987). On this basis, the relatively low gill Na,K ATPase activities in H steelhead and chinook salmon and (to a lesser extent) W steelhead transported and released below Bonneville Dam in late April and early May indicated that these fish were not as physiologically prepared to move into seawater (most fish reach the upper estuary within 1 3 d of release) as were those that were transported several weeks later. Other studies have, however, failed to show a correlation between gill Na,K ATPase activities and electrolyte regulation in seawater, survival in seawater, or smolt-to-adult return rates (Folmar and Dickhoff 1981; Ewing et al. 1985; Richman and Zaugg 1987), thus leading to the conclusion that gill Na,K ATPase activity is insufficient as a sole criterion for hypo-osmoregulatory potential (Boeuf 1993). The consequences of differing gill Na,K ATPase activities for behavior and survival of H and W fish in the estuarine and nearshore marine environments are therefore uncertain, but they are certainly deserving of further investigation. Condition factors of W chinook salmon and steelhead declined during barge transportation to Bonneville Dam, with the largest decreases occurring on those dates during which stress indices were most elevated. Similar changes in condition factor were not seen in H fish. Mean lengths of W fish of both species did not differ between samples taken at Lower Granite and Bonneville dams; changes in condition factor were the result of weight loss during barge transportation. The rapidity (less than 40 h) and magnitude of the weight loss (up to 5% of body weight for W chinook salmon, up to 7.5% for W steelhead) indicates a loss of body water. The body-water content of W chinook salmon and steelhead sampled from barges at Lower Granite Dam may have been elevated as a consequence of exercise and exposure to stressors during collection and loading into barges. Adrenergic effects associated with exercise and stress increase gill perfusion and consequently increase the influx of water across the gills (Haywood et al. 1977; McDonald et al. 1991). Previous studies have reported relatively small increases ( 2.5%) in the whole-body or white muscle water content of stressed (Houston et al. 1971) and exhaustively exercised fish (Milligan and Wood 1986; Wang et al. 1994), presumably because salmonids that are well adapted to freshwater rapidly increase urine output to compensate for increased water influx (McDonald and Milligan 1997). This capability may be compromised during the parr smolt transformation. Increased water absorption from the intestinal lumen (Collie and Bern 1982) and a decreased glomerular filtration rate (Holmes and Stainer 1966) are preadaptive changes that anticipate the need for water uptake and conservation in seawater but which would compound the problem of dealing with water gain during stress in freshwater. Our observations suggest that W fish stressed by collection and loading into barges gained excess water that was subsequently eliminated during barge transportation but that stressed