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1 The Influences of Addition of Minerals to Rearing Water and Smoltification on Selected Blood Parameters of Juvenile Steelhead Trout, Salmo gairdneri Richardson Author(s): Terence M. Bradley and A. W. Rourke Source: Physiological Zoology, Vol. 58, No. 3 (May - Jun., 1985), pp Published by: The University of Chicago Press Stable URL: Accessed: 09/11/ :37 Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. The University of Chicago Press is collaborating with JSTOR to digitize, preserve and extend access to Physiological Zoology.

2 THE INFLUENCES OF ADDITION OF MINERALS TO REARING WATER AND SMOLTIFICATION ON SELECTED BLOOD PARAMETERS OF JUVENILE STEELHEAD TROUT, SALMO GAIRDNERI RICHARDSON' TERENCE M. BRADLEY2 AND A. W. ROURKE3 2Department of Fisheries, Aquaculture and Pathology, University of Rhode Island, Kingston, Rhode Island 02881; and 3Department of Biological Sciences, University of Idaho, Moscow, Idaho (Accepted 11/7/84) Experiments were conducted to determine the influences addition of minerals to rearing water had on selected blood parameters of juvenile steelhead trout, Salmo gairdneri Richardson. Fish that were maintained in water with low mineral concentrations exhibited significant increases in plasma NH' and concomitant decreases in plasma Na+ concentrations. The alterations in the levels of these ions coincided with severe gill swelling and increased mortality. The changes in blood parameters, gill structure, and mortality probably are coincident with early changes associated with smoltification. Fish reared in water supplemented with minerals did not display such changes. It is hypothesized that the low environmental Na+ levels reduced NH' excretion by a Na'/NH' exchange mechanism. INTRODUCTION Teleost fishes are ammonotelic, and as such their major nitrogen excretory product is ammonia. This metabolite is produced primarily by deamination of amino acids in the liver (Vellas and Serfaty 1974), and approximately 98% of plasma ammonia is excreted via the gills (Smith 1929; Payan and Matty 1975). Ammonia excretion may be passive via diffusion of the un-ionized (Fromm and Gillette 1968; Maetz 1973) or ionized forms (Goldstein, Claiborne, and Evans 1982), active by exchange of the ionized form for environmental Na+ (Maetz and Garcia-Romeu 1964; Maetz 1972, 1973; Payan and Matty 1975), or a combination of both (Cameron and Heisler 1983). The ammonotelic nature of salmonids can pose toxicity problems in certain in- 'The authors would like to express their gratitude to the personnel of Dworshak National Fish Hatchery, especially J. Lientz, W. Olson, and J. McClain, for their assistance and cooperation. We thank C. Smith, USFWS, Bozeman, Montana, for the histological work and micrographs and Dr. L. B. Kirschner, Washington State University, Pullman, for his critical review of the manuscript. The authors are indebted to Pauly Waldron for her aid in preparation of the manuscript. T.M.B. expresses his gratitude to Dr. T. L. Meade for his invaluable assistance in the early stages of this work. RIAES contribution no Physiol. Zool. 58(3): C 1985 by The University of Chicago. All rights reserved X/85/ $02.00 tensive culture operations, since high concentrations of ammonia in rearing water can be toxic (Smith and Piper 1975). Long-term exposure to ammonia has been shown to cause major changes in gill structure, including swelling of the secondary lamellae, degeneration of the pillar cell system, and possibly impaired oxygen uptake (Smart 1976). Smith and Piper (1975) found severe hyperplasia of gill epithelium with extensive fusion of lamellae in rainbow trout (Salmo gairdneri) exposed to >25 blg/liter un-ionized NH3 for 6-12 mo. Other deleterious effects include increased water uptake resulting in osmotic imbalance (Lloyd and Orr 1969) and alteration of the oxygen-carrying capacity of the blood (Brockway 1950; Sousa and Meade 1977). Decreases in the concentrations of cerebral ATP, NADH, and succinate after ammonia exposure have also been reported (Arillo et al b). Elevated ammonia concentrations have been shown to increase hepatic lysosomal sensitivity to osmotic shock and to increase total proteolytic activity in the liver (Arillo et al. 1981a). The general consensus for the maximum environmental un-ionized ammonia concentration for rearing salmonids without detrimental effects ranges from 12.5 gg/liter to 20 glg/ liter (EIFAC 1970; Smith and Piper 1975; Wedemeyer 1977; Willingham et al. 1979). The present report examined the influence of environmental mineral concentra- 312

3 CHANGES IN PLASMA CHARACTERISTICS IN SALMO GAIRDNERI 313 tions on selected blood parameters and gill structure in juvenile steelhead trout, Salmo gairdneri Richardson. The studies were conducted at Dworshak National Fish Hatchery (DNFH) located on the North Fork of the Clearwater River in Ahsahka, Idaho. The water feeding this hatchery has an extremely low mineral concentration, and the hatchery has experienced perennial juvenile steelhead trout mortalities. Severe gill swelling usually occurs in mid-december and results in mortalities as high as 25,000 fish/day in a population of 1.5 million fish. The mortalities occur even though the levels of ammonia in the water are extremely low. The hypothesis tested in this paper was that a buildup of endogenous NH'7, not environmental NH3, occurred under low exogenous mineral conditions and was related to the gill changes and fish mortalities. This hypothesis seemed worthy of investigation in light of earlier work suggesting the possibility of a Na'/NH'4 exchange mechanism in certain teleosts, including rainbow trout (Maetz and Garcia-Romeu 1964; Kerstetter, Kirschner, and Rafuse 1970; Kirschner, Greenwald, and Kerstetter 1973; Payan and Matty 1975; Kerstetter and Keeler 1976; Evans 1977; Payan 1978). This report examines the possibility that gill swelling and mortality may result from low exogenous Na+ levels that possibly retard the excretion of ammonia by a Na'/NH' exchange mechanism. MATERIAL AND METHODS Juvenile steelhead trout of the North Fork Clearwater River strain were used in all experiments. The fish were maintained in two systems of 17 ft X 70-ft recirculating Burrows ponds (25 ponds and 35 ponds) at DNFH. Each pond contained approximately 35,000-40,000 fish. The trout were fed an Abernathy formulation dry diet 8-10 times daily. The juvenile steelhead trout were maintained on 90% reused water throughout the study, and temperatures were maintained at C throughout the winter. It was thus possible to rear a mm fish in 1 yr. All reused water was passed through a series of four or six 25 ft X 75 ft X 10-ft polyethylene bead or ring biofiltration beds which maintained average environmental ammonia (NH3-N) and nitrite (N02-N) levels at glg/ml and jlg/ml, respectively. Ammonia concentrations were measured three times per week by direct nesslerization and nitrite concentrations by the sulfanilamide- NEDA method (APHA 1974). The unionized ammonia (NH3) concentration was calculated on the basis of water temperature and ph using an aqueous ammonia equilibrium table (Thurston, Russo, and Emerson 1979). One system of ponds contained control fish. These fish were maintained throughout the course of the study on water from the North Fork of the Clearwater River supplemented with 20 glg/nl Na+, 8 jtg/ ml K', and 30 lg/ml Cl-. Experimental fish were maintained in a separate system of ponds on unsupplemented river water from day 1 (11/10) through day 42. After day 42, these fish were reared in river water supplemented with the same concentrations of minerals as the water feeding the ponds holding the control fish. The juvenile steelhead trout in the experimental and control groups averaged mm and mm, respectively, on day 21. Determination of the effects of mineral supplementation on fish health was based on comparison of the number of mortalities; plasma Na+, K+, and NH' concentrations; light microscopy of gill structures; and gross examination of the fish. From day 1 until day 92 (2/9), trout were randomly dipnetted from the ponds and anesthetized in a 75-gjg/ml solution of tricaine methanesulfonate (MS 222, Argent Chemicals). The total length of each fish was measured to the nearest 0.5 cm, and blood was withdrawn from the caudal vein using a 0.5- or 1.0-cm3 tuberculin syringe with a 25-g X 5/8-inch needle (Pharmaseal). At each collection time, samples of whole blood were collected from 20 experimental and 20 control fish. Each group of 20 samples was pooled into five samples, each of which contained equal aliquots of blood from four individual fish. Heparinized syringes were used to withdraw blood from fish, and the pooled blood was mixed by several inversions. Pooled blood samples were gently

4 314 T. M. BRADLEY AND A. W. ROURKE hand rolled to further ensure mixing and immediately placed on ice. Syringe needles were removed to prevent hemolysis, and the whole blood was gently injected into 350-rl caraway tubes. Samples were centrifuged at 2,000 X g for 15 min at 4 C in a Beckman TJ-6R centrifuge. Plasma and erythrocytes were collected by cutting the tubes at the plasma/erythrocyte interface and placed in separate 0.5-ml plastic vials. Samples for plasma Na+ and K+ determinations were stored in a -25 C freezer. Plasma for NHI assays was maintained at 4 C, and ammonium assays were performed within 3 h after centrifugation. Plasma NH' concentrations were determined with a Sigma Chemicals no. 170 UV assay, and a Radiometer FLM-3 Flame Photometer was used to measure plasma Na+ and K+ concentrations. The data on the blood parameters were statistically examined by two-way analysis of variance. Gill tissue was collected for histological examination at several times. The second gill arch on the right side was excised and placed in Bouin's fixative (Amlacher 1970). After 24 h, the tissue was rinsed in distilled water and transferred to 70% ethanol. Completely dehydrated samples were embedded in paraffin; sections were cut at 5 gim and stained with hematoxylin and eosin-phloxine. At least 10 control and 10 experimental fish were examined at each time point. At least 100 live, moribund, and dead fish were collected daily for gross examination. The gills and mucus were examined for parasites and abnormalities by light microscopy. The internal organs were examined macroscopically for any abnormalities, and bacterial assays were performed (McDaniel 1979). During periods of peak mortality, gill, spleen, kidney, and pyloric caeca samples from over 500 fish were analyzed for infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) (McDaniel 1979). RESULTS Extensive gill swelling in approximately 90% of the experimental fish became apparent on day 27. Feeding activity decreased 2 days later, and the daily ration consumed by all experimental fish decreased from 1,200 lbs on day 29 to 125 lbs on day 44. Figure 1 illustrates the rapid daily mortality increase from 200 to 2,000 fish in a population of approximately 1.5 million. No increased mortalities were observed in the control group (fig. 1) " 1600 S400 S Days FIG. 1.-Mortalities of fish in ponds without mineral addition (0 - *4) and with minerals added (0 - O). Mortalities are presented as total deaths per population per day. I indicates the point of mineral addition to the experimental system.

5 Z CHANGES IN PLASMA CHARACTERISTICS IN SALMO GAIRDNERI 315 Examination of fish for bacterial (Pseudomonas spp., Aeromonas spp., Yersinia ruckeri, and myxobacteria spp.) and viral (IHNV and IPNV) agents revealed that they were not the cause of gill swelling and decreased feeding. The differences between specific control and experimental water parameters are presented in table 1. The mineral levels in the experimental system are typical of past years (Joseph Lientz, personal communication). The un-ionized NH3, NH3-N, and nitrite concentrations for the control and the experimental water are illustrated in figure 2. After addition of minerals, mortalities continued to increase from 1,621 to 2,017 fish on day 47; mortalities then rapidly decreased to less than 400 fish by day 60 (fig. 1). Blood parameters and gill structure also began to return to normal by day 48. Specific changes in gill structure and blood parameters from day I to day 92 are presented in the following sections. GILL STRUCTURE Gill swelling became apparent on day 27 (fig. 3) and was easily determined by gross examination as the filaments protruded beyond the posterior margin of the opercles. Microscopic examination of wet gill tissue (100X and 450X) revealed swelling throughout the filaments, especially in lamellae located in the upper one-third of the filament. Edema and fusion of lamellae, especially at the tip, were prevalent. No abnormalities in gill structure were TABLE 1 RANGE OF SELECTED WATER PARAMETERS IN THE CONTROL AND EXPERIMENTAL PONDS Experimental Control ph Temperature (C) Na+ (sg/ml) K+ ( 1g/ml) Cl- (;g/ml) Ca++ (ug/ml) HCO3 (rg/ml) Dissolved oxygen (mg/liter) NOTE.-Minerals were added to the experimental system on day 43 increasing the Na+, K+, and C1- concentrations to the control levels (20 ;tg/ml Na', 5 Ag/ml K+, and 30 ig/ ml Cl-). E z.03 ON.02 z.01 E z " S5.0- o4.0 z Days C FIG. 2.-Concentrations of N02-N, NH3-N, and NH3 in rearing water. Each point represents the X + SD of three separate readings. (* represents single determinations.) Results are those in control water (O - 0) and experimental water (0 - O). detected in fish from the control group. Addition of minerals to the experimental system on day 43 resulted in a noticeable decrease in gill edema and swelling by day 48. Twenty-seven days after addition of minerals to the water containing the dying fish, no gross microscopic morphological differences between the two groups of fish could be observed. PLASMA NH+ CONCENTRATIONS The plasma NH+ concentrations of control and treatment fish from day 1 to day 92 are presented in figure 4A. Each point represents the mean NH+ concentration + SD for five pooled samples of four fish each. A statistically significant increase (P <.001) in plasma ammonium levels occurred during the period of day 27 to day 40 when experimental values increased from to glg/ml. Control values exhibited a significant decrease (P <.001) during the same

6 : 316 T. M. BRADLEY AND A. W. ROURKE Wr", ii~iiii iiiiiii~iii iiiiii%'i :::::::::: i :-iiii iiii -ii _ owe!:: ~-:~:ciiiiii~ -i_-_-:i i:i~i_----:,i :: iiiiiiiiiiii i;i16ii- Afii i~ i ~ %iiiiiiiii IN iiiii' i-'''' :::-il dxa,. v, Di --i::l aiiiiiiiii"."iiiiii iiiiiiiiii" 'i'"'iiii-i-:i-i~i'iiiijiii01...iii'iiiili a-iiiiii-i: i: i~a~siiiiiiiiiiii'aai i~-iiiia iiii ~ iiiliiiiil~i lop. FIG. 3.-Light micrographs of gill tissue from experimental and control fish. Typical gill lamellae of control fish (a) and experimental fish (b) shown at low (175X) magnification. 10o.o E 8.o z E 2.0- N o J170O z Days FIG. 4.-Levels of plasma NH+ (a) and Na+ (b) in experimental (0 ) and control (0-0) fish. Each point represents the mean of five samples + the standard deviation. I indicates the point of mineral addition to the experimental system. A B period, going from to jig/ml. The time of increase in plasma NH' in experimental fish coincided with the time of the previously mentioned changes in gill structure and increased mortalities. Plasma ammonium concentrations decreased rapidly in experimental fish after the addition of minerals on day 43. By day 47, control and treatment plasma NH' levels were not different at the P <.05 level. PLASMA Na+ CONCENTRATIONS Plasma Na+ concentrations were analyzed in the same samples used for NH' assays, and figure 4B illustrates the changes observed in Na+ concentrations. Plasma Na+ concentrations decreased from 163 t 2 meq/liter on day 27 to meq/ liter on day 40 in experimental fish and increased from to meq/ liter (P <.001) in control fish during the same period. Examination of figures 4A and 4B shows concurrent Na+ and NH'4 changes. A rise in plasma NH' concentrations in the experimental fish coincides

7 CHANGES IN PLASMA CHARACTERISTICS IN SALMO GAIRDNERI 317 with a decline in plasma Na+ concentrations. In control fish, the opposite was seen; plasma NH' decreased as plasma Na+ increased. Figure 4 also shows that the Na+ concentration in experimental fish returned to values comparable to those of control fish within 14 days (day 57) after the addition of environmental minerals. PLASMA K+ LEVELS No significant differences in plasma K+ concentrations were observed between control and experimental fish (fig. 5). A decrease in K+ levels in both groups occurred between day 27 and day 40 (P <.001) and was followed by an increase in K+ levels (P <.01 for both experimental and control fish). DISCUSSION The results provide evidence for excretion of ammonia via a Na+/NHI exchange mechanism in juvenile anadromous Salmo gairdneri. In the experimental system with low external Na+ concentrations, an inverse correlation was observed between plasma Na+ and NH+ concentrations- NH' increased as Na+ decreased (figs. 4A and 4B). The addition of external Na+ (20 pg/ml) resulted in both a decrease in plasma NH' and an increase in plasma Na+. The data indicate that ammonia excretion in juvenile steelhead trout involves an exchange mechanism and not simply passive diffusion of the free form. Addition of 0.87 mm Na+ to the water resulted in a 39% decrease in plasma NH+ concentrations at the next sampling date (fig. 4). These findings are consistent with previous studies indicating the existence of a Na'/NH7 exchange in Carassius auratus (Maetz and Garcia-Romeu 1964; Maetz 1973), S. gairdneri (Kerstetter and Keeler 1976; Payan 1978), and marine teleosts (Evans 1977; Goldstein et al. 1982). The results of the present study indicate a central role for a sodium-dependent excretion of ammonia. Recent studies by Cameron and Heisler (1983) of 1,200-2,000-g S. gairdneri indicated that, under normal resting conditions of low external NH3 and ph, diffusive movement of NH3 appeared to account adequately for ammonia excretion. A Na+/NH7 exchange was indicated during high external am- 3.0 Ll E ZL 2.0 I Days FIG. 5.-Plasma K+ levels in experimental (e - e) and control (0-0) fish. Each point represents the mean of five samples + the standard deviation. I denotes point of mineral addition to experimental rearing water.

8 318 T. M. BRADLEY AND A. W. ROURKE monium treatments. The work presented in this report indicates the presence of such an exchange mechanism with low external NH'. While one cannot pinpoint the reasons for the apparent differences, they could involve the age and size of the fish, the anadromous nature of the fish (smoltification), or the fact that a Na+/NH' exchange is apparent only at certain concentrations of environmental and plasma ions. The recurrence of elevated plasma NH' concentrations, pathological gill changes, and increased mortalities at approximately the same time each year in populations of steelhead trout raised in unsupplemented water may be related to the anadromous nature of these fish. Although changes associated with smoltification in steelhead trout and spring chinook salmon (Oncorhyncus tshawytscha) occur in the spring, recent reports indicate that additional changes occur well before early spring (Bradley and Rourke 1984). The gill swelling and mortalities seen in December may relate to the increased need for a NH' exchange mechanism functioning in the early stages of smoltification. It is possible that one change associated with the earlier periods of smoltification is alteration in protein metabolism. Prior to the time of peak mortalities, plasma NH' may be reutilized by assimilation into amino acids. If reutilization of NH' does occur, excretion rates could be relatively low and the external Na+ concentrations sufficient to support removal by a Na'/NH' exchange. A decrease in NH' reutilization and/or increase in protein deamination that might accompany increased protein turnover at smoltification could increase plasma NHI concentrations. Thus high NH' levels coupled with low environmental Na+ could result in reduced NH+ excretion by a Na+/ NH+ exchange mechanism. Alternatively, changes during smoltification, such as hormonal changes, may change the Na+ affinity of the Na+/NH4- pump. A hormonally induced increase in Km would reduce ammonia excretion at the low external Na+ concentrations. Both alternatives are consistent with the expectation that certain changes associated with smoltification may be influenced by photoperiod and thus occur near the winter solstice, which takes place on December 22 (Zaugg 1982). Previous studies have demonstrated the detrimental effects of sublethal external unionized ammonia concentrations on the gill tissue of salmonids (Burrows 1964; Smith and Piper 1975). The pathological gill changes observed in the experimental fish in this experiment closely resemble those described by Smart (1976) and Smith and Piper (1975). However, the external ammonia concentrations that experimental fish were exposed to were far below the unionized NH3 concentration (12.5 ig/liter) recommended for salmonids (Wedemeyer 1977). The maximum concentration of ammonia to which experimental fish were exposed was only 32% of Wedemeyer's (1977) value, and control fish were exposed to only 6% of this value. Because of the large size of the rearing and water treatment systems and differences in filter bed media, it was impossible to maintain exactly the same external ammonia concentrations for both control and experimental fish. High-pressure cleaning of the filter bed media tends to remove certain bacterial species, causing the peaks in water NH3-N, NH3, and N03-N concentrations observed in figure 2. Nonetheless, both groups were maintained well below recommended limits of ammonia. In experimental fish, the increase in plasma NH' coincided with changes in gill structure and elevated mortalities. Hillaby and Randall (1979) hypothesized that it is either the ionized form or the total ammonia load in the blood that is toxic to fish, not the un-ionized form. The method of ammonia measurement used by Hillaby and Randall resulted in a loss of ammonia gas and underestimation of the true blood ammonia concentrations. Because of this, no comparison can be made between these data and those obtained during the experiment described in this report. It is likely, however, that the increase in plasma NH' concentrations in experimental fish is related to the changes in gill structure. When minerals were added to the experimental system, plasma NHi levels decreased, followed by a decrease in gill swelling and mortalities. This is the first report suggesting that endogenous rather than exogenous ammonia can cause gill changes in vivo.

9 CHANGES IN PLASMA CHARACTERISTICS IN SALMO GAIRDNERI 319 AMERICAN PUBLIC HEALTH ASSOCIATION, AMERICAN WATERWORKS ASSOCIATION AND WATER POL- LUTION CONTROL FEDERATION. In text, APHA Pages in M. J. TARAS, A. E. GREENBERG, R. D. HOAK, and M. C. RAND, eds. Standard methods for the examination of water and wastewater. 13th ed. American Public Health Association, Washington, D.C. AMLACHER, E Textbook of fish diseases. Transl. by D. A. CONROY and R. L. HERMAN. T.F.H. Publications, Neptune City, N.J. 302 pp. ARILLO, A., C. MARGIOCCO, F. MELODIA, and P. MENSI. 198 la. Effects of ammonia on liver lysosomal functionality in Salmo gairdneri Rich. J. Exp. Zool. 218: ARILLO, A., C. MARGIOCCO, F. MELODIA, P. MENSI, and G. SCHENONE. 1981b. Ammonia toxicity in fish: studies on rainbow trout (Salmo gairdneri Rich.) Exotoxicology Environ. Safety 5: BRADLEY, T. M., and A. W. ROURKE An electrophoretic analysis of plasma proteins from juvenile Oncorynchus tshawytscha. J. Fish Biol. 24: BROCKWAY, D. 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