THE EFFECTS OF LOW PH ON SALMONID EMBRYOGENESIS AND VITELLOGENESIS David B. Parker B.Sc.,Simon Fraser University, 1977 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biological Sciences @ David B. Parker 1985 SIMON FRASER UNIVERSITY January, 1985 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL NAME: - David B . Parker DEGREE : Master of Science TITLE OF THESIS: T h e effects of l o w p H on s a l m o n i d e m b r y o g e n e s i s and v i t e l l o g e n e s i s . EXAMINING COMMITTEE: Chairman : Dr. P.C. Oloffs D r . B. A. M c K e o w n , S e n i o r Supervisor -. D r . G .H. ' G e e n D r . T.A. W a t s o n , E n v i r o n m e n t a l S t u d i e s C o - o r d i n a t o r , B.C. H y d r o I 1 " - , & - rell, P u b l i c Examiner 'i I hereby grant to Simon Fraser University the right to lend my thesis, proJect or extended essay ( t h e title o f which i s shown below) to users of the Simon Frassr U n i v e r s i t y L i b r a r y , and t o make p a r t i a l or single copies only f o r such users o r i n response to,a request from t h e other universi9y, o r other educational institution, on i t s own b e h a l f or for one of i t s users, I further agree that permission for mu1 t i p l e copying o f t h i s work f o r scholar I purposes may be granted b y me or the Dean of GraduaPe Studiss. It is understood that copylng o r publication of t h i s work f o r flnancia! g a i n shall not be allowed without my written permission. t i b r a r y o f any T i t l e of Thes i s/Project/Extendsd Essay Author: -: - (signature) - ABSTRACT The effects of low pH water on embryogenesis and vitellogenesis in kokanee and sockeye salmon ( O n c o r h y n c h u s n e r k a ) were investigated. Eggs were exposed to low pH from fertilization to 45 days post-median hatch or to an episodic exposure at pH 4.0. Adult kokanee were also exposed to low pH just prior to ovulation and spawning. Alkali-labile vitellogenin (Vg)-bound phosphates and calcium levels were measured in the plasma of acid exposed rainbow trout (Salrno g a i r d n e r i ). The effect of low pH on calcium metabolism was also investigated by measuring gill c a 2 + - ~ ~ p a and s e i n v i t r o gill osmotic water uptake from rainbow trout exposed i n v i v o . The most sensitive stages of development during chronic or episodic exposure to low pH were early embryonic development and newly-hatched alevins. Incubation of eggs at low pH caused a lower median survival, delayed hatching, higher alevin mortality and reduced the efficiency of yolk conversion to tissue of yolk-sac alevins. Those effects were more pronounced when the eggs were fertilized at low pH. Exposure of sexually mature kokanee salmon to acidified water reduced egg and alevin survival, delayed embryo hatching and decreased the percent hatch. Those effects were more pronounced when their eggs were incubated at low pH. Rainbow trout exposed to pH 5.0 had significantly lower gonadosomatic and hepatosomatic indices compared to control fish, and did not exhibit an increase in plasma Vg-bound iii phosphates indicating that egg production was affected. An increase in environmental calcium concentration significantly affected the plasma vitellogenin levels in fish. No evidence of bone demineralization was found in females exposed to low pH. After 14 days exposure to different levels of pH and environmental calcium, trout exposed to an increased calcium concentration had a lower plasma osmolarity independent of the environmental pH. Gill Ca2+-ATPase activity was not affected by low pH, but was reduced in fish exposed to Ca2+ enriched water. The increase in gill osmotic water permeability in fish exposed to Ca2+-enriched water may have been associated with the increase in osmolarity of the ambient media. The effect of low environmental pH on freshwater survival of salmonids is discussed with particular reference to embryogenesis and vitellogenesis, and the ameliorating effects of water hardness. ACKNOWLEDGEMENTS I would like to extend my sincere appreciation to my supervisor and friend Dr. B.A. McKeown for his continuous support and guidance throughout the duration of my studies. I would also like to thank Dr. T.A. Watson for his help on assay techniques used in this study, and for his advice and friendship through some difficult moments. I wish to extend my gratitude to Dr. G.H. Geen for his advice and helpful criticism of this thesis. To my parents, Jean and Bern, I thank them for their patience and moral support. I wish to thank my wife, Kathleen, for her love, support and encouragement. Funds for this study were provided, in part, through a G.R.E.A.T. award by the Science Council of British Columbia in collaboration with the I.P.S.F.C. and B.C. Hydro. Special thanks to Drs. J.A. Servizi (1.p.S.F.C) and R.G. Ferguson (B.C. ~ y d r o ) for their involvement in this project. I thank Jeff Johansen for his technical assistance. I express special thanks to Bethan Chancey for her love, encouragement and support during those intensive weeks before my defence. TABLE OF CONTENTS .................................................... i i i Acknowledgements ..............................................v ... ~ i s tof Tables .............................................vlll List of Figures ..............................................ix A . General Introduction ....................................... 1 Abstract B . Chapter I-The effects of low pH on egg and alevin survival of kokanee and sockeye salmon. O n c o r h y n c h u s ................................................... 4 I . Introduction .........................................5 I1 . Materials and Methods ..............;................8 Kokanee salmon experiment: ...............8 Sockeye Salmon Egg Experiments ..........13 Water quality analysis ..................14 Statistics ..............................15 I11 . Results ...........................................1 6 Kokanee salmon experiments ..............16 Sockeye salmon experiments ..............24 IV . Discussion .........................................3 6 nerka C . Chapter 11-Effects of pH and/or calcium-enriched freshwater on plasma levels of vitellogenin and Ca2+. and on bone calcium content during exogenous vitellogenesis in rainbow trout ........................44 Introduction I1 . ..............................49 Experimental Fish .......................49 ater rials and Methods Acid Dilution Apparatus ................... 54 Statistical Analyses ....................55 I11 . Results ...........................................57 IV . Discussion .........................................65 Analytical Procedures D . Chapter 111- Effects of pH and/or calcium-enriched freshwater on gill Ca2+-Adenosine triphosphatase ( A T P ~ S activity ~) and osmotic water inflow in rainbow trout ..................................................73 I . Introduction ....................................... :74 I1 . ater rials and Methods ..............................78 Experimental Fish .......................78 Dilution Apparatus ......................7 8 Experimental design .....................7 9 Gill perfusion ..........................80 Gill Preparation for C a 2 + - ~ ~ p a Assay se ..81 c ~ ~ + - A T Assay P ~ s ~.......................8 2 Osmotic Water Inflow ....................8 4 Statistics ..............................8 5 I11 . Results ...........................................8 7 Blood Parameters .......................8 7 ca2+-A~paseActivity ....................8 7 Osmotic Water Inflow ....................90 Plasma Calcium. Hematocrit and Osmolarity .......................9 4 ................... 101 E . Summary ..................................................105 References ..................................................107 Osmotic Water Uptake vii LIST OF TABLES TABLE 1 PAGE Median survival time of kokanee salmon eggs exposed to low pH ................................ 18 Days to hatching, 50% hatch and % hatch kokanee salmon embryos exposed to low pH ................. 20 The efficiency of yolk to tissue conversion in kokanee salmon alevins exposed to low pH ......... 2 2 Length of kokanee alevins after 118 days exposure to low pH ........................................ 23 Median survival and hatching data of sockeye salmon eggs exposed to low pH .................... 27 Hatching data of sockeye salmon embryos exposed to low pH for 24 h .................................. 30 Calcium content of the caudal centra of rainbow trout exposed to low pH and/or calcium-enriched water ........................... 6 3 Female / male plasma Ca2+ ratio of rainbow trout exposed to low pH and/or calcium-enriched water ............................................ 6 4 Blood parameters of rainbow trout after 14 days exposure to low pH and/or calcium-enriched freshwater ....................................... 88 Ca2+-ATPase activity in rainbow trout after 14 days exposure to low pH and/or calcium-enriched water ........................... 8 9 Protein concentrations and C a 2 + - A T P ~ Sin~ rainbow trout after 14 days exposure to low pH and/or calcium-enriched water ........................... 92 viii LIST OF FIGURES FIGURE PAGE Line diagram of the experimental apparatus used to expose sexually maturing kokanee salmon to low pH ............................................... Line diagram of the egg incubation chambers .......... Survival curves of kokanee salmon eggs and alevins exposed to low pH ................................ 11 12 17 Survival curves of sockeye salmon eggs and alevins exposed to low pH ................................ 2 5 Survival curves of sockeye salmon eggs exposed to low pH ........................................ 28 Survival curves of sockeye salmon eggs and alevins exposed to pH 4.0 for 2 4 h ....................... 2 9 Dry tissue / dry yolk tissue ratio of sockeye salmon embryos exposed to low pH ................. 3 3 Dry tissue / dry yolk + tissue ratio of sockeye salmon embryos exposed to pH 4.0 for 24 h ........ 3 4 + Total length of sockeye salmon embryos exposed to low pH ........................................ 3 5 Line diagram of the experimental apparatus used to exposed rainbow trout to low pH and/or increased C a + 2 concentrations .................... 5 3 Plasma alkali-labile phosphorus levels in female rainbow trout exposed to low and/or increased Ca+2 concentrations .............................. 5 9 Plasma calcium ion concentrations in female rainbow trout exposed to low and/or increased C a + 2 concentrations .............................. 6 0 Effect of low pH and/or calcium-enriched water on the GSI and HSI of female rainbow trout .......... 61 Effect of low pH and/or calcium-enriched water on the i n vitro osmotic water inflow of the gills of rainbow trout ................................. 9 3 A. General Introduction Acid deposition is a global problem receiving a great deal of study and public interest with respect to its impacts upon aquatic ecosystems (Likens et al., 1979; Cowling, 1 9 8 2 ) . The basis of acid deposition is an increase in emissions of mineral acids from fossil fuel combustion and other anthropogenic sources (Schindler, 1979; Spencer, 1 9 8 2 ) . These compounds enter aquatic ecosystems as dry deposition or react with oxygen and water vapour in the atmosphere to form sulphuric as well as nitrous and nitric acids, which enter freshwater lakes and streams by precipitation and surface runoff. quat tic systems particularly sensitive to acid deposition are generally soft, poorly-buffered waters low in calcium and magnesium right et a l . , 1980; Henriksen, 1 9 8 2 ) . In northwestern North America, in particular, the west coast of British Columbia, granitic rock and thin soils low in buffering and ion exchange capacity, occupy a large portion of coastal areas ( ~ i e n s ,1 9 8 4 ) . Although these areas are not presently undergoing significant acidification, bedrock geology and water quality data suggest these areas would be sensitive to acid input. Acidification of lakes and streams has resulted in reduced or complete elimination of fish populations in Scandanavian countries and northeastern North America (~eamishand Harvey, 1972; Almer e t Leivistad et al., 1974; Beamish, 1974a; Harvey, 1975; al., 1976; Schofield, 1976; Harvey and Lee, 1982; Schofield, 1 9 8 2 ; ) The reduction in fish populations of acidified lakes has been attributed, in part, to decreased recruitment from a failure of mature fish to spawn (~eamishand Harvey, 1972; Beamish e t al., 1 9 7 5 ) and increased mortality in developing embryos (Peterson et al., 1 9 8 2 ) . While numerous workers have investigated the effects of low pH on embryo and alevin development in salmonid species (Krishna, 1953; Kwain, 1975; Menendez, 1976; Daye and Garside, 1977; Trojnar, 1977; Carrick, 1979; Daye and Garside, 1979; Daye, 1980; Peterson et al., 1980; Brown and Lynam, 1981; Nelson, 19821, none had observed the effects of low pH on Pacific salmon species (Oncorhynchus ) when the present study commenced. No studies have investigated the effects of acidification on salmonids during the vitellogenic period. Many British Columbia waters support salmonid species, either resident or migratory populations, and many of these waters are sensitive to acidification (Swain, 1 9 8 4 ) . Generally, sockeye salmon (Oncorhynchus nerka) migrate further into headwater streams and spend longer in freshwater before spawning, compared with other pacific salmon species. Therefore acidification of freshwater lakes and streams may potentially affect gonadal maturation as well as embryonic development in sockeye salmon. Kokanee salmon are the landlocked or freshwater form of sockeye salmon and consequently, may be even more suscbptible to the impacts of acidification. To elucidate the potential impacts of acidification on Pacific salmon species, I investigated the effects of low pH on both the ovulation and early development of 0. nerka, and on vitellogenesis in rainbow trout (Salmo gairdneri). In this manner, the most sensitive stages of development between exogenous vitellogenesis and the fry stage could be identified. Since it was not possible to obtain kokanee or sockeye salmon in the earlier stages of ovarian development, rainbow trout (S. gairdneri) were selected for use in studies described in Chapter 1 1 . Among salmonids rainbow trout make a good subject to study the effects of low pH on vitellogenesis, as they are considered similar to sockeye salmon in their sensitivity to acid ombo bough, 1982). Calcium uptake is important during the vitellogenic period in fish and any disturbance in calcium metabolism might inhibit vitellogenin production. Therefore, in Chapter I 1 1 the effects of low pH on calcium ion transport and osmotic water uptake in the gills of rainbow trout was investigated. B. Chapter I-The effects of low pH on egg and alevin survival of kokanee and sockeye salmon, Oncorhynchus nerka I. Introduction Low pH may increase fish egg and alevin mortality, reduce the percent hatch, inhibit hatching, and reduce alevin growth (see review by Peterson et al., 1982). Only a few studies have demonstrated these effects on salmonid embryos fertilized at low pH and subsequently incubated at low pH (~enendez,1976; Trojnar, 1977; Carrick, 1979; Brown,1982b). Other studies involving exposure of maturing adult fish, and subsequent fertilization and embryo incubation at low pH have been performed on non-salmonid species (Mount, 1973, fathead minnows; Craig and Baksi, 1977, flagfish; Lee and Gerking, 1980, desert pupfish). The results from studies investigating the effects of low pH on salmonid embryo development are inconsistent and are complicated by differences in prior adult exposure to low pH. Also these studies did not compare all possibilities, ie. eggs fertilized at low pH and incubated at control pH levels versus eggs fertilized at control pH incubated at low pH. Studies on eggs not fertilized at low pH before incubation could underestimate lethal pH levels. Episodic exposure of eggs during fertilization or at later developmental stages, or continuous low pH exposure from fertilization may produce differences in embryo survival. In acidified lakes and streams pH levels are variable, and during periods of high precipitation and snowmelt water bodies having non-toxic pH levels may experience large increases in hydrogen ion concentration. These events generally occur in spring or fall, coinciding with the reproductive events of salmonid fish. Nelson (1982) presented indirect evidence that reduced embryo growth at low pH may result from an inhibition of yolk conversion into adult tissue. The results of several other studies have also suggested reduced yolk conversion in alevins (~rojnar,1977; Brown and Lynam, 1981). Menendez (1976) found that exposure of adult brook trout, (Sal vel i nus f o n t i nal i s), to low pH af fected the survival of the eggs and alevins. Exposure of female fish to low pH has been shown to affect gonadal maturation, resulting in reduced egg production and yolk quality ( ~ o u n t ,1973; Beamish, 1974a; Craig and Baksi, 1977; Lee and Gerking, 1980). These effects are probably a result of the inhibition of yolk production and/or incorporation (Ruby et al. , 1977). However, low pH exposure during final egg maturation and ovulation prior to spawning may also affect embryo survival. In this chapter I investigated the effects of low pH on embryo survival, hatching, and alevin growth and mortality, in eggs obtained from sexually mature kokanee salmon (0.nerka) exposed to low pH during final maturation and ovulation. Sockeye salmon (0.n e r k a ) eggs were exposed to episodic events of low pH or continuously incubated at low pH, with or without fertilization at low p H . Therefore, the most sensitive stages of embryological development in 0. n e r k a exposed to low p H could be determined, as well as the effects of prior adult exposure and fertilization at low p H . IS. ater rials and Methods Kokanee salmon experiment: Kokanee salmon (0.nerka) (77-136 g body weight and 21-24 cm fork length) were caught in a trap net set at the mouth of the Upper Chilliwack River, ~ritishColumbia. Fish were caught during their upstream spawning migration ( ~ u l y28 to Aug. 4) and were transferred to the aquarium facilities at the Sweltzer Creek Laboratory of the International Pacific Salmon Fisheries Commission (1.p.S.F.C.). The fish were held in a 1500-L fiberglass tank supplied with continuously flowing freshwater (hardness, 80-85 mg*L- ' CaCO,; alkalinity, 60.6-68:2 mg*L- ' CaCO,; pH 7.0-7.28; dissolved oxygen, above 90% saturation; temperature 9.0+1.0•‹C). After 7 days, 10 female and 10 male kokanee salmon were transferred to each of the 3 experimental tanks and pH of the water was adjusted to experimental levels over 24 h. The fish were exposed for 24-36 d to the following pH (mean+S.E.): Tank 1 : pH 5.1 f 0.13 Tank 2 : pH 5.6 f 0.46 Tank 3 : pH 7.1 + 0.06 These pH levels were selected to determine the sublethal effects of low pH exposure. During the exposure period female fish were examined routinely to determine their time of ovulation. Approximately 100-150 eggs were collected from mature females in each tank and wet fertilized with sperm from males held at the same pH. Eggs were incubated at pH 4.3+0.1, 5.1k0.08, 5.520.1 and control, 7.6k0.02 (mean + S.E.). Eggs and alevins were removed for histological observation as numbers permitted during the exposure period. Five eggs were removed from each basket on days 3, 17, 33 and 51. Ten alevins were sampled on day 89 and 118, anesthestized in ethyl m-aminobenzoate (MS-222, 50 [email protected]~-')and their weight ( 9 ) and body length (mm) were measured. Eggs and alevins were preserved in either Stockard's solution (50 ml formaldehy.de, 40 ml glacial acetic acid, 60 ml glycerin, 850 ml distilled H,O) or 5% buffered (pH 7.0) formalin (50 ml formaldehyde, 4.0 g sodium phosphate monobasic, 6.5 g sodium phosphate dibasic, made up to 1 L with distilled water). Yolk material was separated from the body tissue of preserved alevins and both parts were dried at 80•‹C for 24 h to determine dry weight. Dead eggs, identified by yolk opaqueness, were counted and removed daily. Alevins were considered dead when their yolk sac turned opaque and their heart stopped beating. Identification of the kokanee egg development stages were made according to those stages outlined by Velson (1980) for sockeye salmon. Eggs were stained with toluene blue after removal of the egg capsule to - provide contrast to the embryonic structures. Fish exp0sur.e tanks Freshwater obtained from the hypolimnetic zone of Cultus Lake flowed into buckets and exited through a plexiglass down tube at approximately 500 Lob-' into the exposure tanks (1500 ~)(~ig. 1). The flow rate allowed for 90% replacement of water in 7 h and a flow of 4.15 L of water for every kg of fish as recommened by Sprague, (1973). The water in the tanks was continuously aerated with compressed air. Acidic pH levels were obtained by adding the required volume of H2S0, stock solutions to the water in the buckets by peristaltic pump. Egg Exposure Chambers Incubation chambers were made of black plexiglass and covered with a black lid ( ~ i g .2). A continuous flow of water into the 'header' tanks was regulated by 'stop cock' valves. To obtain the desired pH in the exposure tanks, H2S0, stock solutions were added to the water in the 'header tanks' by peristaltic pumps. Acidified water exiting from the bottom of the 'header tank' was regulated by 'stop cock' valves to maintain a flow rate of 200 mlomin-' into the incubation chambers. Fig. 1 . Line diagram of the experimental apparatus used to expose sexually maturing kokanee salmon to various pH levels. Water flow f I Peristaltic m Hole size cut to allow a flow of approximately 500 L.h-1 llb ( H2YU41 Stock Somon I U Fig. 2. Line diagram of the egg incubation chambers and experimental apparatus used to expose kokanee and sockeye salmon eggs and alevins to acidified water. Sockeye Salmon Egg Experiments Sockeye salmon eggs were obtained November 1 , 1981, at the Weaver Creek spawning channel, British Columbia. Eggs from four females were mixed together before dividing them in separate groups. Groups of eggs were wet fertilized in water of pH 7.6, 5.5, 5.0, 4.5 and 4.0, and allowed to water harden for 1 h before transporting them to the Sweltzer Creek Laboratory. Eggs fertilized in control water were incubated at pH 4.lf0.1, 4.620.06, 5.0+0.13, 5.720.05 or control, 7.620.02 (mean+S.~.) and eggs fertilized in acidified water were incubated at the pH of fertilization. Two groups of eggs were fertilized at pH 4.0 and 5.0 then subsequently incubated at pH 7.6f0.02. Other groups of eggs were exposed for 24 h at pH 4.0 after 4, 18, 38 and 77 days incubation at control water conditions(pH 7.6). These exposure times corresponded to early blastula development, gastrulation, early organogenesis just after reaching the 'eyed' stage, and 50% hatch respectively. Each group of eggs was separated into two replicates of approximately 250 eggs. Eggs were sampled for histological observation at 24 h, 5 d, 10 d and every 10 d period thereafter. - Eggs sampled during the first 10 days of exposure were preserved in Stockard's solution and eggs sampled 10 days after fertilization were preserved in 5% buffered formalin. Dead eggs and alevins .were identified as previously described and removed daily. Developmental stages of sockeye salmon eggs were identified following Velson (1980). Alevins were anesthetized in MS-222 then blotted dry to determine body weight and length. Dry weight of alevins and yolk sac material were determined as previously described for kokanee salmon. Dilution apparatus To achieve the exposure pH levels, H,SO, was diluted using a proportional diluter constructed similar to that designed by Mount and Brungs, ( 1 9 6 7 ) . Since the exact pH levels required could not be obtained directly from the proportional diluter cells, freshwater regulated by 'stopcock' valves was used to achieve the nominal pH levels. The flow rates into the incubation chambers were maintained at 200 mlemin-'. Chambers were placed in a water bath to maintain a constant temperature in all incubation chambers. Incubation chambers used for the sockeye salmon egg exposures are shown in Fig. 2. W a t e r qua1 i t y anal y s i s Water temperature and pH were measured daily in both kokanee and sockeye salmon egg experiments. Dissolved oxygen, free CO, and hardness were measured periodically during the exposure period. Dissolved oxygen was measured according to the Idometric method using the Azide Modification Technique and was maintained above 90% saturation (American Public Health Association, APHA, 1976). Free CO, was measured by the Titrimetric method (APHA, 1976) and ranged from 4.0 mg [email protected]' at pH 7.6 to 34.6 mg CO,@L-' at pH 4.0. Water hardness was measured by the EDTA Titrimetric method (APHA, 1976). Survival curves and median survival times were calculated according to Cutler-Ederer (1958) using the BMDP (Biomedical Computer programs) data analysis system, Life Tables and Survival functions program (BMDP Statistical Software, 1983). The differences in percentage hatch and alevin survival were compared with their respective controls by testing for differences among proportions (Zar, 1974). The efficiency of yolk conversion to tissue was calculated by dividing the dry tissue weight by the dry yolk plus dry tissue weight. ~fficiency of yolk conversion to tissue and alevin length data were analyzed by using the SPSSX (Statistical Package for the Social sciences) system, SPSSX Manova program (SPSSX User's Guide, 1983). Efficiency group means or alevin length means were compared by analysis of variance (~iner,l971), followed by a Student-Newman Kuels Test to determine the difference between . all possible pairs of means (~50.05). 111. Results Kokanee salmon experiments Adult fish exposed to pH 5.1 were more lethargic and had a build-up of mucus covering their bodies. Thirty percent of these fish died during the exposure period while no mortalities were observed at pH 5.6 and pH 7.1. Mortality was only recorded until the time of egg taking. Two female and three ma.le fish were used to obtain fertilized eggs from fish held at pH 5.1, four female and six male fish from pH 5.6 and seven female and six male fish from pH 7.1. Since sexual-dimorphism was difficult to determine phenotypically, far more males than females were found in the tanks. This, in addition to the fact that some of the females released their eggs in the tanks, accounts for the apparent low number of females used for spawning. All groups had a different survival curve than the control suggesting prior adult exposure, and/or fertilization and incubation at low pH decreases embryo survival. Eggs fertilized and incubated at pH 5.1 and below, independent of prior adult fish exposure, had similar survival curves ( ~ i g .3 ) . ~ortality was high in the early stages of development (Fig. 3 ) with a 50% survival at 2 6 - 3 3 days at pH 5.1 and 2 0 - 2 5 days at pH 4.3 1). (Table Fig. 3. Survival curves of kokanee salmon eggs and alevins exposed to low pH levels from fertilization to 1 1 0 days post-fertilization. Each curve represents one group of eggs. ~ g g sfertilized and incubated obtained from fish exposed to or obtained from fish exposed or obtained from fish exposed at pH to to pH 7 . 6 , 7.1 (control); pH 5 . 6 ; pH 5 . 1 ; Eggs fertilized and incubated obtained from fish exposed to or obtained from fish exposed or obtained from fish exposed at pH to to pH 5 . 5 , Eggs fertilized and incubated obtained from fish exposed to or obtained from fish exposed or obtained from fish exposed at pH to to 7.1; pH 5 . 6 ; pH 5 . 1 ; pH 5 . 1 , 7.1; pH 5 . 6 ; pH 5 . 1 ; Eggs fertilized and incubated at pH 4.3, obtained from fish exposed to pH 7 . 1 ; or obtained from fish exposed to pH 5 . 6 ; - 0 0---0 A- ---- A . 40 60 DAYS 80 Table 1. Median survival time of kokanee salmon eggs incubated at different pH levels (mean+S.E.). *more than 50% survival at day 1 1 0 Group Eggs fertilized and incubated at pH 7.6 obtained from fish exposed to: pH 7.1 (control) pH 5.6 pH 5.1 Eggs fertilized and incubated at pH 5.5 obtained from fish exposed to: pH 7.1 pH 5.6 pH 5.1 Eggs fertilized and incubated at pH 5.1 obtained from fish exposed to: pH 7.1 pH 5.6 pH 5.1 Eggs fertilized and incubated at pH 4.3 obtained from fish exposed to: pH 7.1 pH 5.6 Median survival (days) ----* ----* 84.00 Eggs obtained from fish exposed to pH 5.1 and incubated at pH 5 . 5 or 7.6 had a higher mortality that resulted in a lower median survival time when compared with eggs incubated at the same pH levels •L•E able1 ) . Prior exposure of adult fish to pH 5 . 6 also decreased the survival of kokanee salmon eggs incubated at pH 7.6, compared with eggs obtained from fish exposed to control pH levels incubated at pH 7.6 (Fig. 3). This same effect was n'ot observed in eggs incubated at pH 5.5. Development rate of surviving kokanee salmon embryos was not inhibited by exposure of adult fish to low pH or by incubation of the eggs at low pH. Only eggs fertilized and incubated at pH 5 . 5 and above survived to hatching (Table 2). Eggs fertilized and incubated at pH 5 . 5 irrespective of prior adult fish exposure, had a 7-12 day delay to median hatch compared to all other groups. Exposing parent fish to low pH had no additional effect on the day to median hatch of eggs incubated at pH 5 . 5 . For eggs incubated at control pH levels, prior exposure of adult fish to pH 5.6 and 5.1 resulted in a 6-day delay to first hatch but only a 3-day delay to 50% hatch. A few embryos hatched before 84 days but died emerging. A significantly ( ~ 5 0 . 0 5 ) lower percent hatch was observed in all groups compared with the control (Table 2). The data for eggs fertilized and incubated at pH 5 . 5 are variable, however eggs from adult fish exposed to pH 5.1 showed a marked reduction (13.3%) in the number of eggs hatching. Even by discounting mortality to the beginning of hatching, prior Table 2. Days to hatching, 5 0 % hatch and % hatch of kokanee salmon embryos incubated at different pH levels. Group Days to 1st hatch Days to 5 0 % hatch Hatch* Eggs fertilized and incubated at pH 7 . 6 obtained from fish exposed to: pH 7.1 (control) pH 5 . 6 pH 5.1 78 84 84 84 87 87 100 100 60.4"" Eggs fertilized and incubated at pH 5 . 5 obtained from fish exposed to: pH 7.1 pH 5 . 6 pH 5.1 86 90 95 94 94 96 * ** + % % Hatch+ Alevin survival (%) 76,6 54.9** 41.4** 64.6"" . 36.9** 85.6"" 62.4"" 85.7** 13.3%" 95.6 91.2 87.5 74.4%" 87.7 100.0 % hatch calculated from the number of eggs alive at 1st hatch % hatch from total number Significantly lower than the control at p 1 0 . 0 5 adult exposure affected the hatching success of the embryos •€ able 2). The efficiency of yolk conversion to tissue in developing alevins was not affected by the exposure of adult fish to low pH, providing the eggs were incubated at control pH levels able 3). However, the efficiency was significantly ( ~ ~ 0 . 0 5 ) reduced in alevins hatched from eggs incubated at pH 5.5 obtained from acid exposed adults, compared to alevins from control fish. Fertilization and incubation of eggs at pH 5.5 reduced the efficiency of yolk to tissue conversion in alevins, independent of the pH to which adults were previously exposed (Table 3 ) . Growth (length) was significantly reduced in alevins incubated from fertilization, in all groups exposed to pH 5.5 (Table 4, ~10.05). Exposure of adult fish to pH 5.1 only had a significant effect on the growth of alevins incubated at control pH levels (~10.05). Table 3. The efficiency (tissue/yolk + tissue) of yolk to tissue conversion in kokanee salmon alevins after 1 1 0 days exposure to different pH levels (mean 5 S . E . ) Group Eggs fertilized and incubated at pH 7 . 6 obtained from fish exposed to: pH 7.1 (control) pH 5.6 pH 5.1 Eggs fertilized and incubated at pH 5.5 obtained from fish exposed to: pH 7.1 pH 5.6 pH 5.1 n Tissue/yolk+tissue 10 9 10 0.71820.017 0.74120.034 0.70120.017 a a Table 4. Length of kokanee salmon alevins after 110 days exposure to different pH levels (meanfS.E.). Group Eggs fertilized and incubated at pH 7.6 obtained from fish exposed to: pH 7.1 (control) pH 5.6 pH 5.1 Eggs fertilized and incubated at pH 5.5 obtained from fish exposed to: to pH 7.1 pH 5.6 pH 5.1 n Length (cm) Sockeye salmon experiments A malfunction in the acid-dilution apparatus resulted in variable acidity conditions in groups of eggs incubated at pH 5.0. A dose of very acidic water ( p 3.6) ~ from day 67 to 69 caused mortalities in pH 5.7 groups and the eggs incubated at pH 5.0. The results of groups incubated at pH 5.0 are not reported and mortality curves for groups incubated at pH 5.7 were only calculated to day 65. In all groups, subsamples of dead eggs were examined to determine the percent of unfertilized eggs. This percentage was used to'determine the number of fertilized eggs that died. The corrected values were used to calculate survival. Development rate of the embryos was only affected at day 30 in eggs incubated at pH 5.0 and below. These eggs lagged 2 stages behind all other groups, However, prior to and after day 30, no differences in developmental rate were observed. Episodic exposure of the eggs to pH 4.0 did not affect their developmental rate. The survival curves of embryos incubated at pH 4.0 had the same distribution independent of pH during fertilization ( ~ i g . 4). At pH 4.6 survival of the embryos (eggs and alevins) had two . sensitive periods, a high mortality during early embryonic development (0-20 days) and a sudden decrease in survival just after hatching ( ~ i g .4 ) . Also, those eggs fertilized at pH 4.5 Fig. 4. Survival of sockeye salmon eggs and alevins exposed to low pH levels from fertilization to 110 days post-fertilization. Each curve represents one group of eggs. .~ g g sfertilized at pH 7.6, incubated at pH 7.6 (control); e incubated at pH 4.6; G.....~.... incubated at pH 4.1. A,,,, 0 A Eggs fertilized at pH 5.0 and incubated at pH 7.6. van- ---v Eggs fertilized at pH 4.0 and incubated at pH 7.6. +---+ Eggs fertilized at pH 4.5 and incubated at pH 4.6. 0-0 Eggs fertilized at pH 4.0 and incubated at pH 4.1 . ($--- 0 DAYS and subsequently raised at pH 4.6, initially had a high mortality resulting in 50% survival by 46 days, compared with a median survival of 109 days for eggs fertilized at control pH levels, incubated at pH 4.6 (Table 5). The survival of sockeye salmon eggs fertilized at pH 5.5 and incubated at pH 5.7 had the same survival distribution as the controls (Fig. 5). However, eggs fertilized at pH 7.6 and incubated at 5.7 had a higher mortality with only 25-60% survival at 65 days (Fig. 5 ) . The survival of eggs fertilized at pH 4.0 or 5.0 and incubated at pH 7.6 was similar to the control group to 50 days exposure, at which point mortality increased resulting in a. lower (10%) survival at day 110 (Fig. 4). Exposure of eggs to pH 4.0 for 24 h had the most significant effect on survival when the eggs were exposed during early development (day 4) (Fig. 6). At later stages of development, a 24-h exposure to pH 4.0 had no apparent effect on egg survival. However, hatched embryos exposed to pH 4.0 for 24 h at 77 days (50% hatch) were more lethargic during the acid exposure. No eggs hatched when incubated at pH 4.1 able 5). Eggs fertilized at pH 7.6 and incubated at pH 4.6 had a 12-day delay to 50% hatch (Table 5). The median hatching time was delayed 16 days in eggs fertilized at pH 4.5 and incubated at pH 4.6. The delay in hatching of embryos incubated at pH 5.7 is probably a result of the lethal (pH 3.2) pH exposure at day 67 (see above). The most pronounced effect of the 24 h exposures Table 5. Median survival and hatching data of sockeye salmon eggs exposed to different pH levels (means2S.E.). Group Median survival (days> Time to % % 5 0 % hatch Hatch (a) Hatch (b) (days) Alevin survival % Eggs fertilized a t - p 7~. 6 and incubated at: pH 7 . 6 (control) pH 4 . 6 pH 4.1" Eggs fertilized at pH 5 . 0 and incubated at: pH 7 . 6 Eggs fertilized at pH 4 . 5 and incubated at: pH 4 . 6 Eggs fertilized at p H 4 . 0 and incubated at: pH 7.6 pH 4.1" * no eggs hatched ** significantly different from the control, p 5 0 . 0 5 a % hatch calculated from those eggs surviving to 1st hatch b % hatch calculated from the total number of eggs Fig. 5. Survival of sockeye salmon eggs exposed to low pH levels for 65 days post fertilization. Eggs fertilized and incubated at pH 7.6, control. a -. Eggs fertilized at pH 7.6 and incubated at pH 5.7. A--A Eggs ferti1ize.d at pH 5.5 and incubated at pH 5.7. V----V I I I I 10 20 30 40 DAYS 50 60 Fig. 6. Survival of sockeye salmon eggs and alevins exposed to pH 4.0 for 24 h, once during the 110-day incubation period at pH 7.6. Eggs were continuously exposed to pH 7.6 (control..-.) or exposed to low pH at day 4 ( A A ) , day 18 (v------V), day 38 (C------==---=a), and day 77 i .---.). 60 DAYS Table 6. Percent hatch and alevin survival of sockeye salmon eggs exposed to water of pH 4.0 for 2 4 h (meankS.E.). Exposure Day a b * Time to 50% hatch (days) % % Hatch (a) Hatch (b) Alevin survival % % hatch calculated from those eggs surviving to 1st hatch % hatch calculated from the total number of eggs significantly lower than control, p10.05 on median hatching time was observed on eggs during cleavage (day 4) able 6). Eggs raised at pH 4.6 had a significant reduction in the percentage hatch compared with the controls a able 5). ~ertilizationof the eggs at pH 4.5 further reduced the percentage hatch (37.9%) of eggs incubated at pH 4.6, compared with those eggs fertilized at pH 7.6 and incubated at pH 4.6 able 5). Alevin mortality was significantly greater at pH 4.6 compared with the controls and increased by 15.8% with fertilization of the eggs at low pH able 5, p10.05). pis odic exposure of eggs did not affect the percentage hatch of those eggs surviving to 1st hatch (Table 6). However, a significant reduction (31.6%) in percentage hatch of the total number of eggs was observed when eggs were exposed to pH 4.0 for 24 h on day 4 (pS0.05). Significant decreases in the total percentage hatch were also observed for eggs exposed to pH 4.0 for 24 h at day 18 (5.9%) and 38 (10%)(pS0.05). Alevin mortality was not significantly increased as a result of a 24-h exposure to pH 4.0 at any of the developmental stages. The efficiency of yolk to tissue conversion was inhibited in unhatched embryos exposed to pH 4.6 at day 70 compared to control embryos (Fig. 7). At day 110, alevins still had a significantly lower efficiency than the controls (p10.05). The lower efficiency of yolk to tissue conversion in alevins exposed to pH 5.7 is probably a result of the drop in pH at day 67 (see above). Efficiency was significantly lower in unhatched embryos exposed to pH 4.0 for 24 h at day 4, compared with the controls, however by day 110 no difference was observed ( ~ i g .8 ) . Alevin growth (length) was also significantly reduced by exposure to pH 4.6, independent of the pH at fertilization (p10.05) ( ~ i g .9 ) . Fig. 7. Dry tissue / dry yolk + tissue ratio of sockeye salmon embry*os continuously exposed to low pH levels from fertilization. Each point represents the mean value of 6-8 fish at day 70, and 5 fish at days 90 and 110. Vertical bars represent the S.E. * indicates values significantly different from the control at p10.05. Eggs fertilized and incubated at pH 7.6 (control). Eggs fertilized at pH 7.6 and incubated at pH 5.7. Eggs fertilized at pH 5.5 and incubated at pH 5.7. Eggs fertilized at pH 7.6 and incubated at pH 4.6. Eggs fertilized at pH 4.5 and incubated at pH 4.6. I I I I I 70 80 90 100 110 DAYS Fig. 8. Dry tissue / dry yolk + tissue ratio of sockeye salmon embryos exposed to pH 4.0 for 24 h at different developmental stages. Each point represents the mean value of 6-8 fish at day 70, and 5 fish at days 90 and 110. Vertical bars represent the. S.E. * indicates values significantly different from the control at ~ 5 0 . 0 5 . Control, *a - Eggs exposed to pH 4.0 on day 4, A-- Eggs exposed to pH 4.0 on day 18, v-----IT Eggs exposed to pH 4 . 0 on day 38, a=--=----=-=--0 Eggs exposed to pH 4.0 on day 77, . A . M I I I I 70 80 90 100 DAYS I 110 Fig. 9. Total length of sockeye salmon embryos exposed to low PH levels from fertilization. Each value represents the mean of 1 1 - 14 fish at day 70 and 10 fish at day 90 and 1 1 0 . Vertical bar s represent the S.E. * indicates values significantly di f ferent from the control at p S 0 . 0 5 . Eggs fertilized and incubated at pH 7 . 6 , control. Eggs fertilized at pH 7 . 6 and incubated at pH 5 . 7 . A--A Eggs fertilized at pH 5 . 5 and incubated at pH 5 . 7 . Vg--"-V Eggs fertilized at pH 7 . 6 and incubated at pH 4 . 6 . 0"""""""'C] Eggs fertilized at pH 4 . 5 and incubated at pH 4 . 6 . 0 . 0 DAYS IV. Discussion Few studies have reported on the effects of low pH on early life stages of Pacific salmon (Rombough, 1982; Geen et al., In Press). These studies were either of short duration (10 days) using 'eyed' eggs, alevins and fry (Rombough, 1982) or of chronic exposure (140 days) of eggs exposed from the 'eyed' stage (Geen et al., In Press). The present investigation is the first to report the effects of low pH on Pacific salmon from fertilization to 45 days postmedian hatch. These early stages of embryonic development must be examined before lethal pH levels for Pacific salmon can be determined properly (Rombough, 1982). Decreases in pH to 5.5-and below will have a significant effect on the survival of kokanee and sockeye salmon embryos. The median survival time for sockeye salmon at p~ 4.5 and 4.1 was higher than for kokanee salmon at pH 5.1 and 4.3 respectively, which suggests a difference in tolerance between stocks. Swarts e t al., (1978) found differences in resistance to low pH between different strains of brook trout. The most sensitive stages of development to low pH were cleavage and newly-hatched alevins, with those stages during gastrulation and organogenesis being more tolerant. Survival of kokanee salmon eggs to the 'eyed1-stagewas less than 32% at pH 5.1 and below, and between 18% and 55% at pH 4.6 and below for sockeye salmon. Exposure of sockeye salmon eggs to pH 4.0 during late cleavage (day 4 ) reduced the percent survival to the 'eyed' stage. However, acid exposure for 24 h during gastrulation and organogenesis had no effect. These results are in agreement with other studies which have reported that early embryo development (cleavage), and newly-hatched and buttoned-up alevins are more sensitive to low pH than other stages of development (~ohannsen et al., 1973; Daye and Garside, 1977; Daye and Garside, 1979; Lee and Gerking, 1980; Peterson et al., 1980; Brown and Lynam, 1981; Brown, 1982b). Gjedrem (1980) exposed brown trout eggs to low pH at different stages of development and found an increased survival the later the transfer to low pH was made, suggesting a more graded effect of low pH on embryo mortality. At pH 4.0 most sockeye salmon embryos (64%) were dead before the 'eyed' stage, independent of the pH at fertilization and water hardening. However, at pH 4.6 only 55% survival of acid fertilized eggs to the 'eyed' stage was observed compared with 90% survival of eggs fertilized at pH 7.5 and raised at pH 4.6. Trojnar (1977) reported an estimated survival of 82% to the 'eyed' stage of brook trout eggs fertilized and incubated at pH 4.65. Carrick (1979) observed an increased mortality in Atlantic salmon, sea trout and brown trout eggs fertilized at low pH. Carrick also found salmon eggs fertilized at low pH had an increased mortality (50% within 4 wk) at pH 4.0, in contrast to . the results in the present study which observed high mortalities at pH 4.0 independent of the pH at fertilization. Brown (1982b) found no effect of acid-fertilization at pH 4.5 on the survival of brown trout eggs. Exposure of adult kokanee salmon to pH 5.5 and below just prior to ovulation and spawning reduced egg survival. This effect was more pronounced when the eggs were taken from parents exposed to low pH and subsequently incubated at pH 5.5 and below, suggesting an inhibitory effect of low pH during final egg maturation (Fig. 3). Kennedy (1980) also observed that previous exposure of adult lake trout to low pH affected gametogenesis and fertilization causing significant mortality in the early stages of embryonic development. Fertilization and incubation of kokanee salmon eggs at pH 5.5 and below increased embryo mortality. Sockeye salmon eggs fertilized and incubated at pH 4.5 and 4.6 respectively, had a lower median survival time than eggs fertilized at control pH and subsequently incubated at pH 4.6. However, the effect of low pH on fertilization and water hardening may be only temporary unless the eggs are subsequently incubated at low pH, since the survival of sockeye salmon eggs fertilized at pH 4.0 or 5.0 and incubated at control pH was only reduced by 10%. These results may also infer that a time-dependent exposure is involved. Eggs acid-fertilized at pH 4.0 and 5.0 were transferred to control pH levels within 3 h. Peterson and Martin-Robichaud (1982) found 100% mortality of newly fertilized ova of Atlantic salmon after - 8 h exposure to pH 4.0. Exposure of parent fish to low pH delayed hatching and reduced the percent hatch of kokanee salmon eggs irrespective of the incubation pH. Eggs exposed to low pH, independent of parental exposure, also had a delay in hatching time. Delayed hatching of eggs from acid exposed parents has been observed for brook trout (~enendez,1 9 7 6 ) ~fathead minnows ( ~ o u n t ,1973) and desert pupfish ( ~ e eand Gerking, 1980). The delay in time to median hatch in sockeye salmon eggs fertilized at pH 4.5 and subsequently incubated at pH 4.6 is in contrast to other findings. Peterson et a1 . ( 1980) found no delay in hatching of Atlantic salmon eggs reared at low pH from fertilization. Whether these eggs were fertilized at low pH or transferred to low pH water after fertilization' is not reported by the authors. Daye and Garside (1979) observed no delay to median hatch for Atlantic salmon eggs fertilized at control conditions and incubated at low pH. However, Peterson et al. (1980) observed delayed hatching in eggs exposed to low pH from the 'eyed' stage. A delay in time to median hatch and a reduced hatchability of eggs exposed to low pH from the 'eyed' stage have been reported in other studies Nelson, 1982; Geen et al ., In Press). warts et Peterson al., 1978; et al ., (1980) and Nelson (1982) suggested that adaptation during early development may have occurred to allow for normal hatching. Adaptation may occur in eggs during early development, however results in the present study suggest hatching is inhibited if fertilization and water hardening take place at low pH. The results of the sockeye salmon experiments show a median lethal pH level of 4.6 for a 50% hatch of eggs fertilized in control water and raised at low pH. wain (1975) reported a median lethal pH value of 4.75 at 10•‹c for a 50% hatch of rainbow trout eggs under similar fertilization and incubation conditions. However, in the present study, eggs raised from fertilization at pH 4.6 had a much lower percent hatch (16%) than eggs placed in low pH water after fertilization. Carrick (1979) also found approximately the same percent hatch (5%) for ~tlanticsalmon eggs fertilized and raised at pH 4.5. These results suggest that studies in which eggs are not fertilized at low pH may underestimate the lethal pH levels. No eggs survived to hatching at pH 4.0 irrespective of the pH level during fertilization and water hardening. These results are similar to those obtained by Peterson et al. (1980) who found hatching of Atlantic salmon eggs was prevented at pH 4-0. Kwain (1975) observed no survival of rainbow trout embryos below pH 4.49 regardless of temperature. Exposure of parent kokanee salmon to low pH had a negative effect on the survival of newly-hatched alevins subsequently raised at control pH levels. The mechanism by which parental exposure to low pH in the later stages of egg maturation affects the survival of eggs and alevins is not known. The results in the present study do not allow for interpretation of the mechanisms involved. Menendez (1976) also observed an increase in alevin mortality as a result of parental exposure at pH 5.09. The kokanee alevin mortality data were variable, but do suggest a pH effect on eggs raised at pH 5.5 and below. Menendez (1976) found 56% alevin mortality at pH 5.09 in alevins hatched from eggs obtained from control fish. The mortality of sockeye salmon alevins was greater for eggs that had been fertilized and raised at low pH, than eggs fertilized at control pH levels and incubated at low pH. By 30 days post median (50%) hatch, alevin mortality was 68-83% at pH 4.6. Rombough (1983) found newly hatched sockeye salmon alevins and buttoned-up alevins had ten-day L C s 0 (Sprague, 1973) pH values of 4.57 and 5.02 respectively. Geen e t al. (1n press) also observed significant mortality in newly hatched chinook salmon alevins at pH 5.0 and below. The efficiency of yolk conversion to tissue and the growth (length) of kokanee and sockeye yolk-sac alevins was reduced at pH 5.5 and 4.6, respectively (Table 3 and ~ i g .7). These results confirm assumptions made by Nelson (1982) that reduced growth at low pH may result from an inhibition of yolk conversion into tissue. Nelson (1982) found a reduced concentration of water-soluble proteins in acid exposed rainbow trout alevins, which suggested an inhibition of the conversion of yolk-sac material to tissue. This disruption of normal protein anabolism could be a result of 1 ) direct deactivation of protein synthesis by H'; 2 ) an inhibition of large yolk sac proteins into low molecular weight fragments or 3) a reduced absorption and transport of these fragments elso son 1982). Reduced growth and efficiency rate in sockeye salmon alevins exposed to pH 5.7 was a result of the drop in pH which occurred at day 67. However, these results do show that a 2-day exposure to a lethal pH value of 3.2 will result in reduced growth of surviving alevins raised at pH 5.7. Lethal pH values (pH 4.5-5.0) reported in this study are slightly lower than pH values reported in the literature for salmonids of similar sensitivity to low pH. In part, the difference is attributed to the fact that eggs were fertilized in acidified water in the present study, compared with many other studies that fertilized eggs at control pH and then transferred the eggs to low pH. Also, increased free CO, concentrations probably accounted for the lower .lethal pH values, especially in newly hatched alevins. Lloyd and Jordan ( 1 9 6 4 ) observed lower resistance times of rainbow trout at pH values above 4.0 when dissolved C02 was above 20 ppm. However, embryos and alevins may be exposed to increased free CO, concentrations during incubation in redds. The concentration of CO, in stream-bed interstitial water has been observed to increase with increasing depth of the substrate (Williams and Hynes, 1974). Acidification of streams and rivers may further increase interstitial water CO, concentrations , which may be more important to egg survival than pH. In summary, the results show that water of pH 5.0 or less will significantly reduce embryo survival. Further, exposure of adult fish to pH 5.6 prior to ovulation and spawning, results in reduced egg survival. These results are in general agreement with survival data for other teleost fish reported in the literature. Delayed hatching, inhibition of hatching, and reduced efficiency of yolk to tissue conversion as well as other observed effects of low pH on developing embryos, may be due to an inhibition of enzyme systems involved in embryonic development, and/or a reduced egg viability resulting from an inhibition of the processes involved in egg production and maturation in female fish exposed to low pH. C. Chapter 11-Effects of pH and/or calcium-enriched freshwater on plasma levels of vitellogenin and c a 2 + , and on bone calcium content during exogenous vitellogenesis in rainbow trout I. Introduction Four physiological periods have been identified in the reproductive cycle of female rainbow trout, S. gairdneri, by van Bohemen et al., (1981). These are the previtellogenic period arch-~pril), a period of endogenous vitellogenesis ( ~ a y - ~ u l y ) when the oocytes synthesize their own yolk, a period of exogenous vitellogenesis (~ugust-~ecember) when the oocytes take up vitellogenin from the blood and a period of ovulation and spawning. Exogenous vitellogenesis is a period where oviparous vertebrates incorporate large amounts of yolk proteins into developing oocytes. This involves the production of yolk proteins in the liver which are secreted into the bloodstream, transported to the ovary and incorporated into the oocytes by micropinocytosis (wallace, 1978; van Bohemen et al., 1981). Histological studies of the liver show dramatic structural changes and an increase in cell size during exogenous vitellogenesis (van Bohemen et al., 1981). Vitellogenin (Pan et al., 1969) in teleosts has been identified as a female-specific lipophosphoprotein molecule that binds calcium ( ~ o r iet a1 ., 1979; whitehead et a1 ., 1983). During the period of exogenous vitellogenesis blood phosphorus and calcium ion levels, which are part of the vitellogenin complex, rise considerably (whitehead el a1 ., 1983). The extra calcium required for the protein complexes is taken up from the external environment and/or internally from demineralized bone (Fleming et al., 1964; Ma, 1976). Consequently, calcium regulation in fish also becomes important during ovarian maturation. Loss of fish populations from acidified lakes is partially caused by a decreased recruitment due to a failure of mature fish to spawn ( ~ l m e re t al., 1974; Beamish, 1974a; Beamish and Harvey, 1972; Beamish et al, 1975). Abnormal development of the ovaries has been observed in lake herring and rock bass caught in acidified lakes (Beamish, 1974a). Ovarian maturation and spawning may be the most sensitive of all physiological processes related to the survival of fish populations in acidified lakes (Beamish, 1976). Laboratory studies have shown that fish exposed to low pH have a reduced frequency of spawning, lower number of eggs per female and a decreased number of viable eggs (~rishna,1953; Mount, 1973; Menendez, 1976; Craig and Baksi, 1977; Lee and Gerking, 1980). The quality of eggs in fish exposed to low pH is also reduced. Mount (19731, for example, found the eggs of fathead minnows exposed to pH 5.9 lacked turgidity and had fragile shells. Egg quality, as determined by opaqueness of the eggs and yolk diameter which are associated with exosmosis and protein coagulation, was reduced in desert pupfish (Cyprinodon nevadensis n.) exposed to acidified water ( ~ e eand Gerking, 1980). A reduction in the ability of oocytes to form mature eggs due to an inhibition of secondary yolk deposition was observed in the ovaries of flagfish exposed to pH 6.0 and below (Ruby et al., 1977). A reduction in yolk deposition in growing oocytes may not only be a result of an inhibition of yolk uptake at the ovary but also an inhibition of vitellogenin production and secretion in the liver. Although no studies have measured plasma vitellogenin levels in sexually maturing female fish exposed to acidified water, abnormally low serum calcium levels in female fish during ovarian maturation (Beamish et 1975) may al., indicate inhibition of vitellogenin synthesis. Low pH exposure may affect calcium metabolism resulting in low serum calcium levels during ovarian maturation which consequently effect regulator factors involved in vitellogenesis. . Field (Wright and Snekvik, 1978) and laboratory (McDonald et al, 1980; Brown, 1982b; McWilliams, 1982; Nelson, 1982) studies have shown that environmental calcium concentration affects the survival and physiological process in fish exposed to acidified water. Therefore, environmental calcium concentration in acidified waters may ameliorate the inhibitory effects of increased H + concentration on ovarian development. vitellogenesis in salmonids may be affected at sublethal pH levels (pH 5.0) which could result in complete inhibition of egg production or an increase in the production of abnormal eggs resulting in high mortalities during embryonic development. In the present study the effects of acidified water and different . environmental calcium ion concentrations on the plasma vitellogenin and calcium ion levels in salmonid fish during the period of exogenous vitellogenesis were investigated. Bone calcium content and the gonadosomatic and hepatosomatic indices were determined in trout after low pH exposure during the vitellogenic period. 11. Materials and Methods E x p e r i m e n t a l Fish Three-year old autumn spawning rainbow trout, S. g a i r d n e r i , approximately 250-600 g body weight and 28-38 cm in fork length were obtained from Sun Valley Trout Farm, Mission, British Columbia. The fish were kept in a 2000-L fiberglass tank supplied with flow through dechlorinated tap water (Ca2+ 0.045-0.069 6.3-6.50) mM, Na+ 0.024 mM, Hardness 5.2 mg*~-'CaCO,,pH with the temperature ranging from 11"-15•‹C over the 6 week acclimation period. The photoperiod was changed weekly to simulate a natural photoperiod (~ug.-Nov., 50" Latitude, ~ancouver). The fish were fed dried pellets (Moore and clark, Laconner, Washington) daily a d l i b i t u m . Fish that were not feeding or did not appear healthy were removed from the holding tank and were not used for experimental purposes. After acclimation, fish were randomly selected and anesthetized in a buffered (sodium bicarbonate, NaHCO,) solution of ethyl m-aminobenzoate (MS-222, 50 mg*L-'1. Approximately 0.5-0.7 mL of blood was taken by caudal vascular puncture with an ammonium heparinized (10,000 i.u.1 syringe. Body weight and fork length were recorded at the beginning and end of the subsequent exposure period and each fish was marked with coloured beads attached with nylon line immediately anterior to the dorsal fin. The fish were revived in fresh water and exposed for 64-70 days to one of the following solutions. a) Group I. Dechlorinated water, pH 6.66k0.13 Ca2+=0.0520+0.006 mM, number (N) of fish=lO b) Group 11. Dechlorinated water, pH 6.66k0.12 ~a~+=1.52+0.31mM, N=8 c) Group 111. Dechlorinated water, pH 5.03k0.39 ~a~+=0.052080.006 mM, N=8 d) Group IV. Dechlorinated water, pH 5.1420.34, Ca2+=1.55k0.27 mM, N=8 At two week intervals the fish were anesthetized and blood was sampled by caudal vascular puncture. After 70 days exposure, the fish were killed by severing the spinal cord just posterior to the medulla oblongata, and the gonads and liver were excised for wet weight measurements. Blood samples were analyzed for haematocrit, alkaline-labile phosphate and Ca2+ concentrations. Caudal centra were excised from the caudal peduncle immediately above the insertion point of the last anal fin ray and frozen at -20•‹C for analysis of total calcium content. A c i d Di 1 ut i o n A p p a r a t u s The dilution apparatus received dechlorinated water into a 230-L fiberglass holding tank ( ~ i g1 0 ) . A submersible pump transferred the water to a 100- L plexiglass 'header' tank equipped with an overflow pipe. The incoming water flow was regulated by a 'rotoflow' valve and the pH was adjusted to pH 6.70 with potassium hydroxide. The water exited from the bottom of the header tank through 1/2" tygon tubing into one of the four treatment tanks (800 L). Water flow into the exposure tanks was regulated by 'rotoflow' valves at 2 em in-'. The water in the tanks was continuously aerated with compressed air filtered through a charcoal air filter. This apparatus met the conditions suggested by Sprague (1973) for holding fish. There was more than a liter of water per 10 g of fish with a flow rate slightly less than one liter per minute for each kilogram.of fish resulting in approximately 75% replacement of water in 8 h. Low pH and/or high calcium solutions were prepared by the addition of sulfuric acid (H2S0,) and/or calcium nitrate Ca(NO,), respectively, to the water flowing from the 'header' tank. Both H2S04 and Ca(~0,), were added to the water by a peristaltic pump (~anostat)from stock solutions through 5/32" tygon tubing connected to a 13-gauge needle. The needle was inserted into the water line by a rubber serum stopper connected to a Nalgene 'Y' junction. A float switch located in the header tank was connected to the peristaltic pump which would shut off the pump if the water in the header tank dropped too low. This ensured that the fish did not receive a concentrated solution of . H + and/or Ca2+ if the water pressure dropped. Water temperature, pH and ca2+ concentration were monitored daily. Dissolved oxygen and free CO, were analyzed weekly during the exposure period. The calcium concentration of the water was measured by the E D T A titrimetric method ( A P H A , 1 9 7 6 ) standardized with an atomic absorption spectrophotometer. Oxygen was measured by an iodometric method using an azide modification technique and remained above 80% saturation ( A P H A , 1 9 7 6 ) . Free CO, was measured by the titrimetric method and never exceeded 2.0 mg CO,@L-' ( A P H A , 1 9 7 6 ) . The temperature ranged from 15•‹C at the beginning of the experiment to 9 ' ~after 70 days. Fig. 10. Line diagram of the experimental apparatus used to expose rainbow trout to low pH and/or increased C a + 2 concentrations. Inflow Overflow 'Rotoflow' 1 Air Air 1 Acid or Calcium A n a l yt i c a l P r o c e d u r e s After collection of the blood, each sample was placed in a 1.5 mL polypropylene micro-centrifuge tube and the plasma was collected by centrifugation. The plasma was removed and stored in 400-)JL polypropylene micro-centrifuge tubes at -20•‹C. Hematocrit was determined by centrifuging a separate sample of the blood in 100-L heparinized hematocrit tubes. Plasma Ca2+ was measured by atomic absorption spectrophotometry (Pye Unican, SP 191), using lanthanum chloride to reduce phosphate interference. ~emale/male plasma calcium ratios were calculated by dividing the plasma Ca2+ concentration of each female by the mean plasma ca2+ concentration of males from all groups. The mean plasma ca2+ concentration of males in the experiment was used since no significant difference in plasma Ca2+ concentration was found in males among the groups (p>0.05). Plasma vitellogenin content was estimated indirectly by measuring the alkali-labile protein phosphorus concentration. This technique, although not as accurate as many other time consuming methods which measure vitellogenin directly (Plack e t a2 , 1971 ; Goedmakers and Verboom, 1974; Idler et a1 , 1979; van Bohemen e t al, 1 9 8 1 ) ~is a reliable indicator of the levels of vitellogenin in the plasma of fish (Emmerson and Peterson, 1976; Nath and Sundararaj, 1981). Alkali-labile protein was prepared from duplicate samples according to the technique of Wallace and Jared (1968) with minor changes in the volumes used. The amount of inorganic phosphate in the solution was determined by the method of Martin and Doty ( 1 9 4 9 ) ~using 1/5 the recommended volumes. Phosphate standards were made up from reagent grade potassium dihydrogen phosphate (KH~PO,),dried at 1 1 0 • ‹ ~in , double distilled water. . Total Ca2+ concentration of the caudal centra was determined by the method described by Wendelaar Bonga and Lammers (1982). Two caudal centra from each fish were cleaned in 1.0 N KOH for 2 h and subsequently rinsed 3 times in 100% ethanol. Samples were dried at 90•‹cfor 18h to.determine dry weight and then dissolved in 3.0 mL ION nitric acid (HNO,) for 2 h. A 0.5 mL aliquot of the HNO, plus bone solution was diluted with 4.5 mL of double distilled water. Total Ca2+ concentration was measured with 50 rL duplicate samples by atomic absorption spectrophotometry. Glassware used to prepare chemical solutions was acid washed and glassware used to make solutions for c a 2 + analysis was also rinsed in 10 mM EDTA. Statistical Analyses All statistical analyses were performed by using the SPSSX data analysis system, SPSSX Manova program (SPSSX User's Guide,l983). A multivariate analysis of variance was used to test whether plasma Ca2+ or alkali-labile phosphate measurements differed among the treatment groups (Winer, 1971). Homogeneity of group variances was tested using Bartlet's test and normality by ~olmogorov-Smirnov goodness of fit test. ~eterogeneityof the alkali-labile phosphate group variances was reduced by applying a ln(x + 1) transformation. Group means of the total calcium content of the caudal centra, were compared by an analysis of variance . Gonadosomatic index (GSI) was calculated by dividing the gonad weight by somatic weight minus the gonad weight, (body wt. - gonad wt.) multiplied by 100. The hepatosomatic index (HSI) was obtained by dividing the liver weight by somatic weight multiplied by 100. To determine the effects of low pH and/or calcium on GSI and HSI, the data were transformed to arcsine values and compared by an analysis of variance, followed by a Student-Newman-Keuls test to determine the differences among the group means ( ~ a r ,1974). Significance was accepted at the 95% confidence level with trends being discussed at the 90% confidence level. 111. Results One fish in each group, except for the control, died during the experimental period. Acidification of the water could not be attributed to the cause of death of these fish. Aggressive activity between the fish may have caused some mortalities. Exposure of fish to low pH and/or calcium-enriched freshwater for 65-70 days had no significant effect on hematocrit (p>0.05). The ovulation period for the stock of fish used in the present experiment was approximately October 15-November 30. Plasma calcium concentrations measured in female fish one month prior to the beginning of- the experiment, were already elevated in some fish. Because of this asynchronous timing of gonadal maturation, those fish with initially high plasma vitellogenin content and elevated plasma calcium levels were not included in the results. By excluding these early maturing fish, a comparison could be made between more synchronized fish. One female fish from each of group I and I 1 were excluded and two fish from group 1 1 1 . No fish were excluded from group IV. These fish were left in the tanks throughout the exposure period to keep the relative number of fish per tank the same. The range of plasma Vg-bound phosphates reported here (0.15 - 17.64,umolam~-' plasma) is close to those values (0.49 - 25.61 ~ m o l a m ~ - lreported ) for rainbow trout by other investigators (Whitehead et a l . , 1980, 1983) and within the range for (0.35 29.81 pmol*m~-')catfish, Het e r o p n e u s t e s f o s s i l i s , ( ~ a t hand Sundararaj, 1981). Only trace amounts of total protein-bound phosphates were detected in male fish. In female fish, environmental calcium had a greater effect on Vg-bound phosphate production than pH. Plasma Vg-bound phosphate levels in females exposed to calcium-enriched water were not significantly different at day 0. However, significantly higher levels were observed at day 28 and 65-70 ( ~ i g .lla, pS0.05). The results of the multivariate tests, although not significant, did suggest that low pH has a tendency to inhibit Vg-bound phosphate production. This trend is shown in Fig. llb, where Vg-bound phosphate levels are lower at day 28 and 65-70. The significance (~10.05) of the univariate tests at day 28 and 65-70 further support the observation that low pH had some influence on plasma vitellogenin levels. Plasma calcium ion concentrations in female fish followed a similar pattern as the alkali-labile phosphate levels (Fig. 12a and 12b). As expected increasing the environmental Ca2+ concentration had a greater effect on plasma c a 2 + concentration than decreasing the pH level. Plasma Ca2+ concentrations were significantly (~10.05)higher in fish exposed to calcium-enriched water at day 28 and 65-70. Exposure of females to pH 5.03 (group 111) was associated with a significant (PS0.05) reduction in the GSI at day 65-70, compared with the other groups (Fig. 13). Fig. 1 1 . Plasma alkali-labile phosphorus levels in female rainbow trout ( N = 6 ) exposed to (a) high C a 2 + ( rn , 1.52-1.55 mM) or low C a Z + ( , 0.052 mM) concentrations independent of pH and (b) low pH ( o , pH 5.0-5.1) or control pH levels ( rn , pH 6 . 7 ) , independent of the Ca2' concentration, for 65-70 days. * indicates a significant difference between the groups at p10.05 by analysis of variance. Thin vertical lines represent the standard error ( S . E . ) . Fig. 12. Plasma calcium ion concentration in female rainbow trout ( ~ = 6 ' exposed ) to (a) high C a 2 + ( , 1.52-1.55 m ~ or ) low c a 2 + ( 13 , 0.052 m ~ concentrations ) independent of pH and (b) low pH ( o , pH 5.0-5.1) or control pH levels ( , pH 6.7), independent of the Ca2+ concentration, for 65-70 days. * indicates a significant difference between the groups at p10.05 and t indicates a significant difference between groups at psO.10 by analysis of variance. Thin vertical lines represent the standard error (S.E.). Fig. 13. Effect of low pH and/or calcium-enriched water on the gonadosomatic aand hepatosomatic indices of sexually maturing female rainbow trout ( ~ = 3 )after 65-70 days exposure. Thin vertical lines represent the standard deviation (S.D.). Standard errors not wide enough to be shown graphically are omitted. * indicates values significantly different from the control (Group I). I-pH 6.66, [Ca2+]=0.052 mM; II- pH 6.66, [Ca2+]=1.520mM; III-pH 5.0, [Ca2+]=0.052 mM; IV-pH 5.1, [Ca2+]=1.550 mM. FEMALES FEMALES However, calcium-enrichment completely ameliorated the effects of low pH exposure on gonad size since no significant change in the GSI was observed compared with the control (p>0.05). The HSI of females exposed to acidified water was significantly lower than the HSI of all other groups ( ~ i g .13, pS0.05). HSI and GSI of male fish were not statistically tested due to the small sample size ( N = 1 ) in group 111. Bone calcium content of females from the different groups was not significantly different able 7, pS0.05). Male fish exposed to calcium-enriched freshwater (groups I1 and IV combined) showed a higher (~10.05)bone calcium content in comparison to fish exposed to freshwater (groups I and 11, combined). This difference may have been biased by the low sample size in group I11 (Table 7). However, by combining males and females, the analysis showed a significant effect of environmental calcium on bone calcium content (Table 7, pS0.05). Low pH caused a significant reduction in the female/male plasma c a 2 + ratio while an increased environmental calcium concentration resulted in a significantly higher ratio (Table 8, p10.05). Although non-significant, fish exposed to acidified, low calcium ion freshwater (group 111) tended to have a lower female/male plasma calcium ratio compared with all other groups (Table 8 ) . - Table 7: The effect of pH and/or environmental calcium on the calcium content of the caudal centra in rainbow trout (mean + S.E.). (I) pH 6.66, C a + 2 0.052 mM ( 1 1 ) pH 6.66, C a + 2 1.520 mM ( 1 1 1 ) pH 5.03, C a + 2 0.052 mM ( I V ) pH 5.14, C a + 2 1.550 mM Group Sex n Calcium (mEq*100 g - * includes all fish in the tanks b+d>a+c, p10.05 ' bone) Table 8: Ratio of female/male plasma ~ a concentration + ~ for fish exposed to the following solutions S.E.). for 65-70 days (mean ( I ) pH 6.66, ~ a 0.052 + ~ mM ( 1 1 ) pH 6.66, Ca+' 1.520 mM ( 1 1 1 ) pH 5.03, Ca+' 0.052 mM (IV) pH 5.14, C a + 2 1.550 mM + Group n ~emale/male ratio IV. Discussion In the present study, female trout exposed to acidified water (pH 5.03) had a significantly lower GSI than control fish, indicating egg production was inhibited by low pH. his agrees with earlier studies that have shown a reduced egg production in fathead minnows (Pimephales ~ r o r n e l a s ) exposed to pH 5.9 and below ( ~ o u n t ,1 9 7 3 ) ~flagfish (~ordanellafloridae) exposed to pH 6.0 and below (Craig and Baksi, 1977) and in desert pupfish (Cyprinodon n. nevadensis) exposed to pH 7.0 and below ( ~ e eand Gerking, 1980). Conversely, Menendez (1976) found that total egg production was not affected when female brook trout (Salvelinus fontinalis) were exposed to pH 5.0 although the viability of eggs was reduced significantly. Field observations have shown that white suckers (Catastomus commersoni) failed to reproduce in acidified lakes because they did not reach spawning condition (Beamish and Harvey, 1972). Beamish (1974a) observed abnormal development of the ovaries in lake herring from an acidified lake (pH 4.4 4.9). These fish had only a portion of their ovaries containing developing eggs, which were abnormal in texture and size, indicating a reduction in yolk deposition. Ruby et al. (1977) observed that primary oocyte development and secondary yolk deposition in flagfish ovaries were impaired at pH 6.0 and below. Desert pupfish exposed to pH 5.0 and 5.5 also had fewer developing oocytes in their ovaries than control fish, although the difference was not significant (Lee and Gerking, 1980). Peterson et a1 (1982) suggested that if low pH caused a disturbance in ribonucleic acid (RNA) or protein synthesis during early oocyte development, then this may have caused an inhibition of primary and secondary yolk deposition later on. It appears from the above information that the effects of acid stress on physiological parameters associated with gonadal maturation are related to an inhibition of oocyte development and incorporation of vitellogenin into the ovary. However, yolk deposition in the ovaries may also be inhibited.by a reduction in vitellogenin synthesis by the liver. Rainbow trout exposed to pH 5.0 - 5.1 (group, I11 and IV) for 65-70 days, for example, had a lower rate of Vg-bound phosphate production (Fig. llb). Fish held in acidified water (group 111, pH 5.0) were the only fish not showing a rise in plasma Vg-bound phosphates during the experimental period. In addition, the significantly lower HSI in trout exposed to pH 5.03 (group 111) also suggests that vitellogenin synthesis in the liver was inhibited. Van Bohemen et al. (1981) found that the HSI increased during exogenous vitellogenesis in rainbow trout. If yolk deposition in the ovaries was impaired rather than vitellogenin production in the liver, then plasma Vg-bound . phosphates might be expected to increase. Yaron e t a1 (1977) observed exaggerated levels of calcium and protein in ovariectomized and estradiol-injected fish. Therefore, the lower GSI in trout exposed to pH 5.03 (Group 111) may be a result of low Vg-bound phosphate production by the liver as well as some inhibition of yolk deposition in the ovaries. Environmental calcium concentration may limit vitellogenin production since fish exposed to low environmental calcium had significantly lower plasma Vg-bound phosphate levels. This might be explained on the premise that a higher concentration of environmental calcium promotes a "healthier" fish and that CaZ+ required during vitellogenesis is obtained with less energy expenditure from calcium rich water compared with low calcium water and/or the loss of calcium from the body .is greater in low calcium environments. In a survey of 700 acidified lakes, Wright and Snekvik (1978) reported that calcium content of the water was as important to the fish as was pH. The calcium concentration of acidified waters influences the survival times of fish as well as the number of species present ( ~ r o w n ,1982a). Calcium is bound to the plasma Vg-bound phosphates in teleosts and increases in concentration during exogenous vitellogenesis (Oguri and Takada, 1967; Yaron et a l l 1977; Whitehead e t all 1980), thus inhibition of calcium metabolism in female fish during gonadal maturation may disrupt normal vitellogenin production in the liver or impair yolk deposition at the ovary. Female trout exposed to acidified water (pH 5.03, . group 111) only reached a female/male plasma calcium ratio of 1.15 while for females in the other groups the ratio was greater than 1.4. Beamish e t a1 (1975) found that suckers experiencing reproductive failure in acidified lakes had [email protected]/male serum calcium ratios below 1.4. Abnormally low plasma calcium ratios or levels in female fish during vitellogenesis observed in the present study and reported from field observations by Beamish e t a1 (1975), may be due to an inhibition of active ca2+ transport. Ma (1976) suggested that the gills were the site of calcium uptake since gill Ca2+-ATPase enzyme activity increased during growth and sexual maturation in rainbow trout. However, Ma's experiments se between also showed no difference in gill C a 2 + - ~ ~ p a activity spawning female and male rainbow trout. That was explained by suggesting that extra calcium required by females is most likely obtained from the external environment during growth and sexual maturation, and stored until required for vitellogenesis. An alternate explanation for the lack of any difference in C a 2 + - ~ T P a s eactivity between spawning female and male rainbow trout may be due to the time period at which the measurements were made. Ma (1976) measured C a 2 + - ~ ~ p a enzyme se activity in trout and salmon during the spawning period, and not the period of exogenous vitellogenesis and subsequent increase in plasma calcium levels. Vitellogenin levels in female rainbow trout peak during late vitellogenesis and decrease during ovulation and spawning (van Bohemen e t a l , 1981; Scott and Sumpter, 1983). Lockhurt and Lutz, (1979) found that female white suckers did not have an elevated serum calcium concentration in comparison with males during early gonadal maturation and spawning. However, these authors did find female white suckers to have elevated serum calcium levels during ovarian development. Therefore, even though Ma (1976) reported no difference in Ca2+-ATPase activity between spawning female and male trout, an increase in C a 2 + - ~ T ~ a activity se may occur during the preceding period of exogenous vitellogenesis. Further, if extra calcium taken up from the external environment by female fish is stored internally, then differences in bone calcium levels between sexually mature females and males should be evident. In this study, however, no significant difference in bone (caudal central .calcium concentrations were observed between females during ovarian maturation and male fish. Fraser and Harvey (1982) found that centra calcium concentrations in white suckers did not differ between sex or age, although the stage of sexual maturity of the fish was not reported. No evidence of bone demineralization was found in female fish exposed to low pH or low pH and calcium-enriched water. Therefore, these low calcium levels observed during vitellogenesis in rainbow trout are probably a result of an increased loss of calcium or an inhibition of calcium uptake. These results are in agreement with Lockhurt and Lutz (1977) but contrast to those reported by Fraser and Harvey (1982). Lockhurt . and Eutz (1977) found no indication of any loss of calcium from rib bones, muscle, scales or skin from white suckers caught in acidified or non-acidified lakes. Fraser and Harvey ( 1 9 8 2 ) on ~ the other hand, found the calcium concentration of backbone 8- centra was reduced 16% in fish from acidified lakes relative to fish from non-acidified lakes. These authors explained their findings in comparison with Lockhurt and Lutz (1977) as the difference in analyzing rib bones versus centra. Fraser and Harvey (1982) found ribs and opercular bones were not decalcified in acid stressed fish but hypurals, trunk and caudal centra were. Rainbow trout in the present study may not have been exposed to low pH water long enough to cause any significant changes in bone decalcification or show any reduced calcification during growth. Field observations. showing fish with low bone calcium content ( ~ r a s e rand Harvey, 1982) and skeletal deformities (~eamishe t a!, 19751, are probably a result of an inhibition of calcium uptake and calcification during growth since Nelson (1982) found ossificaion of skeletal parts in developing rainbow trout alevins was inhibited by low Rainbow trout exposed to pH 5.0 fed with less activity than fish in non-acidified water and calcium-enriched acidified or non-acidified water. In general, intestinal calcium uptake in teleost fish is minimal (pang et ai, 1978). However, some of the extra calcium required by female fish during ovarian maturation may come from dietary sources. The effects of food availability . on gonadal maturation in fish has been reported by several authors (see Lam, 1983). Scott, (1962) reported a delayed maturation and reduced egg number in rainbow trout fed on a restricted diet. Therefore, abnormal development of the ovaries A in acid stressed fish could be due to; 1) reduced feeding activity of the fish and/or, 2 ) reduced food resources caused directly or indirectly by acidification. Beamish (1974b) found no relationship between food availability, and reduced growth and increased mortality of white suckers in two acidified lakes. However, in a laboratory study, Beamish (1972) observed that white suckers exposed to pH 5.0 and below had a reduced feeding intensity. Yellow perch in acidified lakes had a reduced growth rate in older age classes which may have been due to food supply and a change in prey species ( ~ y a nand Harvey, .1981). Some physiological parameters and biochemical changes occur in fish during starvation such as anemia, low blood sugar and reduced serum protein, which were not evident in fish sampled from acidified lakes (Lockhart and Lutz, 1977). However, these data are from surviving fish and may represent a more tolerant group of individuals within the population. In summary, the results of this study suggest that low pH exposure of sexually maturing rainbow trout will inhibit exogenous vitellogenesis. Inhibition of vitellogenin production and/or incorporation of yolk proteins into developing oocytes, may result in non-viable eggs and a failure of mature fish to spawn. High embryo mortalities due to poor egg viability will result in a decreased recruitment and a reduced rainbow trout population. Environmental calcium has a ameliorating infuence on the effects of low pH exposure, since fish held in low pH, hard water, did not show an inhibition of vitellogenin production. In fact, increasing the environmental C a + 2 concentration significantly enhanced plasma vitellogenin levels above those levels in fish held in soft water. Calcium complexes with vitellogenin and therefore an inhibitory effect of low pH on calcium metabolism may be partially responsible for the reduced vitellogenin levels reported here. An inhibition of Ca2+ transport mechanisms may impair the ability of female fish to obtain extra calcium required during ovarian maturation as well as maintain normal calcium homoestasis. ( D. Chapter 1 1 1 - Effects of pH and/or calcium-enriched freshwater on gill Ca2+-Adenosine triphosphatase ( A ~ ~ a s eactivity ) and osmotic water inflow in rainbow trout I. Introduction The gills of teleost fish are the main site of monovalent ion and some divalent ion (Ca2+)transport, and osmotic water movement (Potts, 1977; McDonald, 1983). These interrelated processes are partially responsible for maintenance of ionic and osmotic equilibria. The effect of acidification on plasma osmolarity, ionic balance and other physiological parameters have been reviewed by several authors (Fromm, 1980; Spry et al., 1981; McDonald, 1983). The reduction in plasma Na' of acid-exposed fish is a result of an increased Na' efflux (packer and Dunson, 1970; McWilliams and Potts, 1978; McDonald and Wood, 1981) and an inhibition of Na+ influx ( ~ c ~ i l l i a mand s Potts, 1978; McWilliams, 1980; McDonald and Wood, 1981). Increased Na' efflux may be due to an increased gill permeability caused by the removal of surface bound Ca2+ by H + (~cwilliams,1983). A decrease in Na' uptake may be a result of an inhibition of gill Na+-K'-ATPase enzyme activity (~ohnstonee t al., 1983). Plasma c a 2 + concentrations do not vary after exposure of rainbow trout to acid water conditions (Leivestad et al, 1976; McDonald and Wood, 1981). McDonald and Wood (1981) found that ca2+ ion fluxes across the gills of trout exposed to low pH did not change, although urinary Ca2+ losses increased. 'Beamish et a1 (1975) observed that female suckers in acidified lakes did not develop an elevated plasma C a + 2 concentration normally associated with gonadal maturation. Also, female rainbow trout exposed to low pH water had a female/male plasma calcium ratio lower than fish held at control pH levels (Chapter 11). This suggests that c a 2 + regulation in female fish had probably been impaired. The chloride cells in the gills of freshwater rainbow trout have been identified as the site of calcium uptake (Payan et al, 1981). A Ca2+ dependent ATPase enzyme located in the gill plasma membranes may be involved in c a 2 + transport (Ma. e t al, 1974; Fenwick, 1979; Ho and Chan, 1980). Doneen (1981) described two kinetic forms of C a 2 + - ~ T ~ a s one e , with a high substrate affinity (assayed at 100 p M Ca2+) and the other with a low substrate affinity (assayed at 4 mM Ca2+). Flik et a1 (1983) have recently re-evaluated the characteristics of C a 2 + - ~ T ~ a activity se and found that Ca2+-activated ATP hydrolysis in the plasma membranes of eel gills partially results from non-specific alkaline phosphatase activities. These authors also suggest that more than one type of enzyme is involved in transepithelial Ca2+-transport and that the high affinity C a 2 + - ~ T ~ a activity se may represent calcium ion transport. The effect of low pH on active Ca2+ transport . mechanisms in fish gills has not been studied. Most studies concerned with acid toxicity on fish iono regulation have dealt with the movement of monovalent ions in fish exposed to acutely lethal pH levels. Since ionic problems in fish are partially an indirect consequence of their gill permeability to water ( ~ o t t s ,19771, the effects of low pH on ionic regulation may be partially due to changes in osmotic permeability. Structural changes in the gill epithelium would also result in changes in gill permeability to water as well as ions. This study represents the first to investigate the effects of low pH on the gill osmotic water uptake of rainbow trout. Calcium ions in the external media have been shown to affect the permeability of the gills to monovalent ions (Potts and Fleming, 1971; Isaia and Masoni, 1976; McWilliams, 1982) and water movement ( ~ o t t sand Fleming, 1970; Ogawa, 1974; 1saia and Masoni, 1976; Wendelaar Bonga and Van Der Meij, 1981). Therefore, increased survival of fish in acidified water of increased Ca2+ levels (Brown, 1981, 1982a, 1982b11983) may be due to the ameliorating effects of Ca2+ on ion losses across the gills. The inability of fish exposed to low pH to regulate plasma calcium levels during gonadal maturation, might be due to an inhibition of active ca2+ transport processes or ion imbalances caused by changes in gill permeability to water. The long-term regulation of plasma calcium may be more important than the short-term loss of ions at more lethal pH levels (Spry 1981). et al., C Although plasma Ca2+ concentrations in fish exposed to low pH do not change markedly, transport processes may be inhibited, with Ca2+ levels being maintained by such mechanisms as bone demineralization at an added energy expenditure. Changes in gill osmctic permeability due to exposure to low pH may also result in ionic disturbances. However, the environmental calcium concentration may be an important factar in determining to what extent low pH effects ionic and osmotic regulation. Therefore, this study was designed to investigate the effects of low pH ) high and low external Ca2+ concentrations on levels ( ~ ~ 2 5 . 0at Ca2+-ATPase activity presumably reflective of active calcium transport, and osmotic water uptake in the gills of rainbow trout ( S a l m o g a i rdneri ) . 11. Materials and Methods Experimental F i s h Rainbow trout (S. gairdneri, 200-300 g) were purchased from Sun Valley Trout Farm, Mission, British Columbia and transported to the aquarium facilities at Simon Fraser University. The fish were held in 2000-L fiberglass tanks supplied with continuously flowing, aerated and dechlorinated water (~ardness5.2 mgaL- ' as CaCO,; Ca2+ 0.045-0.069 mM; Na' 0.024 mM; dissolved oxygen, 80% saturation; pH 6.3-6.5; 14+0.5•‹C). The fish were fed Oregon moist pellets daily a d libitum, and kept under a simulated natural photoperiod (~uly-~ugust, 50' Latitude, Vancouver) for at least one month prior to experimental use. Fish were not fed 48 h prior to experimentation or throughout the duration of the experimental period. Di 1 ut i o n Apparat u s The dilution apparatus used to obtain the desired Ca2+ and pH levels is shown in Fig 10 (Chapter 111, except the final solutions from the header tank drained into 104-L black plexiglass exposure chambers. Chambers were subdivided into six 14-L compartments with a series of holes through each compartment wall to allow for a complete circulation of the water. Incoming water flowed into a 18.5-L compartment in front of the six holding compartments then through a series of holes in each holding compartment and out the back drain holes. The flow rate was maintained at 5 Lemin-'. Airstones with compressed air bubbling through them were placed in the front compartment and maintained oxygen concentrations above 80% saturation. Water temperature, pH and Ca2+ concentration were recorded daily. Calcium ion concentrations were measured by the EDTA titrimetric method (American Public Health Association, APHA, 1976). Oxygen concentrations were measured periodically during the exposure period according to the azide modification technique (APHA, 1976). Free CO, was measured by the titrimetric method (APHA, 1976) and never exceeded 2.0 mg*~-'. E x p e r i m e n t a1 d e s i g n Before the start of the experiment fish were selected at random and anaesthetized in a buffered (NaHCO,) solution of MS-222 (50.0 rng*~"). Weight and fork length were recorded and a sample of blood was collected by caudal puncture with an ammonium heparinized (Sigma) needle. An aliquot of blood was transferred to two 100 ul heparinized hematocrit tubes and centrifuged for 3 min to obtain hematocrit values. The remaining blood was centrifuged for 5 min in a 1.5 mL polypropylene micro-centrifuge tube and the plasma collected and stored in -r1 polypropylene tubes at -20'~. 400 Following anaesthesia, fish were placed in exposure tanks for 14 days (photoperiod 1 1 L : 13 D). Plasma Ca2+ was measured by atomic absorption spectrophometry (Pye Unican, SP 191), using lanthanum chloride to reduce phosphate interference. Osmolality was measured with a vapour pressure osmometer (model 5100 Four groups of fish ( ~ = 7 ) were exposed to the following solutions: a) Group I Ca2+ 0.052 mM, pH 6.62 b) Group I1 Ca2+ 1.50 mM, pH 6.63 c) Group I11 Ca2+ 0.052 mM, pH 5.00 d) Group IV ca2+ 1.50 mM, pH 5.10 . Calcium concentrations were increased from 0.0520 mM to 1.50 mM in groups I1 and IV to represent the calcium content of lakes of intermediate hardness (100-150 m g @ ~ - 'as CaCO,). Gill P e r f u s i o n After the 14-day exposure period, the fish were anaesthetized in a 50 [email protected]' solution of MS-222 buffered ( p ~ 7.0) with NaHCO, and their weight and fork length recorded. During the gill perfusion, a continuous flow of an aerated 20 m9.L-l solution of MS-222 was maintained over the gills of the fish. A longtitudinal incision was made on the ventral surface to expose the heart and bulbus arteriosus. Blood was collected by heart puncture with an ammonium heparinized (sigma) needle. Hematocrit values were recorded and the plasma collected and stored as previously described. A cannula was inserted into'the bulbous artericsus and tied off around the ventral aorta with surgical thread immediately anterior to the branchial arteries. To drain the blood during the perfusion, the caudal peduncle was cut off and an incision made in the ventricle. The gills were perfused with heparinized (0.1%) teleost saline ( ~ a C 1 ,7.25 g*L-'; KC1, 0.38 g * ~ - ' ;CaCl,, 0.162 g*L-'.; MgS04*7H20, 0.23 g*L-'; NaHC03, 1.0 g*L-'.; NaH2P0,*H,0, 0.36 g * ~ - ' ;Glucose 1.0 g*L-'; Wolf, 1963) at a rate of 5.1 mlamin-'. with a 50 cc syringe connected to a syringe pump (model 341 A, Sage Instruments). Gill P r e p a r a t i o n f o r C a 2 + - A T P a s e A s s a y Gills were excised, rinsed in ice cold saline (0.9% NaC1) and blotted dry. The gill lamellae were trimmed from the gill arches and placed in a preweighed 10-mL beaker containing 5.0 mL of homogenizing media (0.25 M sucrose, 5.0 rnM EDTA, 10 mM imidazole buffer) and weighed to determine gill filament weight. Homogenizing medium was added to the gill filaments to provide a 10% w:v preparation. The gill filaments were homogenized with a glass mortar and pestle (15 passes). The homogenate was centrifuged in a Sorvall superspeed RC2-B centrifuge at 1,000g for 10 min to sediment nuclei. The supernatant was decanted off and centrifuged at 25,000g for 20 min to sediment the mitochondria. The supernatant was then placed into ultracentrifuge tubes and centrifuged at 35,000g for 1 hour at 3OC (~eckmanL2-65B) to obtain the heavy microsomal fraction. The microsomal pellet was resuspended in 5mM Tris-HC1 buffer, 0.5 mM imidazole buffered to pH 7.8 at 25% weight by volume of the gill filament weight. This enzyme homogenate was placed on ice and used immediately for the C a 2 + - A T ~ a s e assay. C a 2 + - A T P a s e Assay Enzyme activity was measured by the Caz+-dependent adenosine triphosphate (ATP) hydrolysis of inorganic phosphate. To measure the low affinity ATPase activity, 0.1 mL of enzyme suspension was mixed with 1.8 mL 3mM CaCl,, 20 mM Tris-HC1 ( p ~ 8.0) and allowed to preincubate for 10 min at 20'~. The reaction was started by the addition of 0.1 mL vanadate-free sodium ATP at a final concentration of 5 mM and incubated for 20 rnin at 20•‹C. The reaction was stopped by the addition of 0.5 mL cold 30% trichloroacetic acid. Reaction vials were centrifuged at 900 g for 5 min to isolate precipitated proteins. High affinity ATPase activity was measured by using 100)(M CaCl,, 20 mM ~ris-HC1,(pH 8.0) incubation media. Assay procedures followed those described above for the low affinity enzyme. All glassware was acid washed in chromerge for 24 h and rinsed with 10 mM ethylenediamine-tetraacetate (EDTA). Double distilled ca2+-free water was used to make up the incubation media and all other chemical solutions used in the assay. Blanks contained either 1.9 mL 3 mM CaC1, or 1.9 mL 1 0 0 p M CaC1, and 0.1 mL Na,ATP. All measurements were done in triplicate for each individual fish. Inorganic phosphate was measured in 1.0 mL aliquots as described by Peterson (1978) using subsequent modifications by Doneen (1981). The concentration and quantity of sodium dodecyl sulfate (sDS) was changed as well as the addition of p-methylaminophenol sulfate (ELON) in place of aminonaphtholsulfonic acid (ANSA). Briefly, the 1.0 mL sample was added to 1.0 mL of 2.5% ammonium molybdate in 4 N HC1, followed by the addition of 1.0 mL 8% SDS and 0.5 mL 2% ELON in 5% sodium sulfate as the reducing agent (Lebel et al., 1978). The volume was then increased to 5 mL with double distilled water, vortexed and read on a perkin-~lmer spectrophotometer olema man 55) at 700 nm after standing for 30 minutes at 20•‹C. Standard phosphate solutions were prepared by diluting a stock solution of KH,PO,. The standards and blanks were treated the same as the sample solutions. All glassware was acid washed and rinsed in double distilled water. This method provided good colour stability and retention of linearity in the range of tihe standard curve. c a 2 + - ~ ~ p a activity se was expressed as pmol Piamg protein-'ah-'. Total protein in the enzyme homogenate was measured by a modification of the Albro (1975) technique. The procedure involved the addition of 50 uL of enzyme homogenate to 2 mL of 0.25 N NaOH. After a 10 rnin preincubation, 1.0 mL of alkaline copper solution (10% Na,CO,, 0.1% sodium tartrate, 0.05% cupric sulfate) was added followed by another 10 rnin incubation. Four mL 0.111 N phenol was then added, incubated at 55•‹C for 5 rnin and then cooled rapidly in ice water for 1 min. The samples were then read at 650 nm within 30 rnin on a Perkin-Elmer spectrophotometer. Bovine serum albumin (Sigma) was used as the standard protein solution over the range of 10 pg*m~' to 200 )~g.mL- '. Osmotic Water Inflow Following the gill perfusion, the first two anterior gill arches on the right side of the fish and the first anterior gill arch on the left side were excised and rinsed in ice cold teleost saline (0.9% ~aC1). In v i t r o osmotic water uptake by the gills was determined by the procedure outlined by Ogawa et al. (19731, Ogawa (19741, and Lock (1979). The isolated gills were placed in 20 ml of oxygenated teleost saline (270 mOsm) and incubated for 20 rnin at 2 0 " ~ .After the 20 rnin incubation period the gills were blotted dry for 15 sec and their weight determined. The gills were then placed in double-distilled water ( 2 0 " ~ )for 15, 30 and 45 min. They were weighed before and after each 15 rnin time interval, After the final weighing the arches were dried at 1 1 0 " ~for 24 h to determine dry weight. St at i s t i c s A11 statistical analyses were performed by using the SPSSX data analysis system, SPSSX Manova program (SPSSX User's Guide, 1983). Before performing statistical analyses, all variables were checked for homogeneity of variances (Bartlett's test) and normality. A factorial analysis of variance was used to test for differences among the group means for each of the following parameters, Ca2+-ATPase activity, osmolarity, plasma Ca2+ and microsomal protein (Winer, 1971). An arcsine transformation was performed on hematocrit data before comparing the differences among group means by analysis of variance. An analysis of covariance was used to test whether osmotic water inflow rates differed among the four treatment groups ( ~ i n e r ,1971; Neter and Wasserman, 1974; SPSSX User's Guide, 1983). The effect that the initial water content of the gills had on water retention and osmotic inflow rates was tested and corrected for in the analysis, as a covariate. There was no significant difference (p10.05) in dry weight of the gills between the groups. Significance was accepted at the 95% level (~10.05). The dry weight of the gills was not significantly different (p>0.05) among the groups, therefore differences in osmotic water influx were not affected by the size of the gills. After a 20 min preincubation in a media of known osmotic value, the osmotic water influx during the incubation periods in distilled water should, therefore, be dependent upon the condition of the gill membrane as well a s the osmotic gradient rather than the mass of gill tissue. 111. Results B l o o d Parameters No significant differences in the plasma Ca2+ levels of the blood were observed between the groups (Table 9 , p>0.05). Exposure of fish to low pH and/or calcium-enriched freshwater had no significant effect on hematocrit. Fish exposed to calcium-enriched freshwater ( 1.5 mM Ca2+) exhibited a significant reduction in plasma osmolarity in comparison with fish exposed to water of 0.052 mM ca2+ (p10.05). Exposure of fish to pH 5..0-5.1, regardless of the environmental calcium concentration, caused a slight but non-significant decrease in plasma osmolarity Ca2+-ATPase Act i v i able 9). t y In fish exposed to pH 5.0 or 5.1, specific activity of the low affinity (assayed at 3 mM c a 2 + ) C a 2 + - ~ T P a s eenzyme was not significantly different from fish exposed to control pH levels (p>0.05). Low-affinity ca2'-~Tpase activity was significantly reduced in fish exposed to calcium-enriched freshwater, regardless of pH able 10, p10.05). There were no significant effects of low pH and/or increased ambient calcium Table 9: Blood parameters of rainbow trout after 14 days exposure to water of different pH and/or calcium concentration (mean k S.E.). ( I ) pH 6.62, C a 2 + 0.052 mM; (11) pH 6.63, C a 2 + 1.50 mM; ( 1 1 1 ) pH 5.00, C a 2 + 0.052 mM; (IV) pH 5.10, C a 2 + 1.50 mM; Group Hematocrits % Osmolarity (mOsm*~g-') CaZ+ (mM) Table 10: C a + 2 - ~ T P a s eactivity in rainbow trout after exposure to solutions of different pH and/or calcium concentration (mean k S . E . ) . ( I ) pH 6.62, C a 2 + 0.052 mM; (11) pH 6.63, C a 2 + 1.50 mM; (111) pH 5.00, C a 2 + 0.052 mM; ( I V ) pH 5.10, C a 2 + 1.50 mM; Group N C a 2 + - ~ ~ p a sactivity e (umol Piemg-'proteinah-') [ C a + 2 ]3mM [ C a 2 + ] 100 yM concentration on the specific activity of the high affinity (assayed at 100 p M C a 2 + ) Ca2+-ATPase enzymes (Table 10). High-affinity C a 2 + - ~ ~ p a activities se showed a trend similar to the low- affinity enzyme in fish exposed to calcium-enriched freshwater. Although not significant, the quantity of protein recovered in the heavy microsomal fraction of the gills showed a tendency (P=0.077) to be higher in fish exposed to calcium-enriched water (Table 11). The total enzyme activity in the gills, expressed as pmol Pioh-log-' gill filament tissue, showed no significant difference in fish exposed to low pH and/or calcium-enriched freshwater able 11). A slight increase in the gill microsomal protein without a simultaneous increase in total enzyme activity may suggest a synthesis of proteins other than those from enzymes involved in ATP hydrolysis. Osmot i c V a t e r I n f l ow Exposure of fish to calcium-enriched freshwater significantly enhanced the rate of osmotic water influx, regardless of pH (Fig. 14, ~10.05). Although pH did not significantly affect the rate of weight increase of the gills of group I11 fish (low C a 2 + , pH 5.0) the graph showed a slightly . higher weight increase than the control group (Fig. 14, p>0.05). The graph of group I11 is linear and coincides with the graphs of the calcium-enriched freshwater groups (11 and IV) to the 30 min incubation point. After the 30 min interval the rate of weight increase drops off and water uptake is no longer linear. This sudden drop in the linear uptake of water might indicate a change in the structural integrity of the gill membranes (Lock, 1979) from exposure to low pH and low calcium. Table 1 1 : Protein concentrations and low affinity ~ a + ~ - ~ ~ ~ a s e (PM ~ i e g - 'gill filament tissue) in rainbow trout after 14 days exposure to different pH and/or calcium ion concentrations (mean + S.E.). (I) pH 6.62, C a 2 + 0 . 0 5 2 mM; (11) pH 6.63, C a 2 + 1.50 mM; (111) pH 5.00, C a 2 + 0 . 0 5 2 mM; (IV) pH 5.10, C a 2 + 1.50 mM; Group N Microsomal Protein ( ~ g e m g'- gill tissue) CaZ+-ATPase activity (pmol [email protected]@g-l gill) Fig. 14. Effect of low pH and/or calcium-enriched water on the i n v i t r o osmotic water inflow of the gills of rainbow trout exposed i n v i v o . At the time intervals, each point represents the mean gill wet weight (adjusted by the covariate, initial water content) of all fish in each group. TIME (min.) IV. Discussion Plasma Cal ci urn, Hemat ocri t and Osmol ari t y The ability of rainbow trout to maintain normal plasma Ca2+ concentrations during exposure to acidified water (pH 5.0 or 5.1) in the present study, agrees with the findings of Leivestad and Muniz, ( 1 9 7 6 ) and McDonald and Wood, (1981). However, McKeown et al., ( 1 984) recently reported some .fluctuation in plasma calcium ion levels in rainbow trout exposed to pH 4.9 and a tendency for lower plasma calcium ion levels in suckers (Cat ost omus macrocheil us). reared at pH 4.9 and 5.6 relative to control fish. Other studies have also reported some tendency for plasma calcium ion levels to fall (McDonald et al., 1980) or fluctuate (Saunders et a1 ., 1983) in fish exposed to low pH freshwater. Plasma ca2+ concentrations in fish exposed to calcium-enriched freshwater did not change relative to fish exposed to low environmental calcium concentrations. These results are in agreement with observations made by other authors on fish exposed to increased external calcium concentrations (~meharaand Oguri, 1978; McDonald et al., 1980; Wendelaar Bonga and Van Der Meij, 1980; Shephard, 1981). The absence of a change in hematocrit of acid ( p 5.0-5.1) ~ exposed fish suggests: 1 ) no significant hematological disturbances occurred and/or 2)the exposure period was long enough and/or the pH was high enough for fish to recover from initial disturbances. Hematocrit values reported in the literature for fish exposed to low pH are variable. These discrepancies may have been dependent on exposure time of the fish, species differences, field versus laboratory measurements as well as the H + concentration of the ambient environment. Chronic exposure of fish to water of pHS4.0 (Neville, 1979) or acute exposure to pH<5.0 (McDonald e t al., 1980) caused an increase in hematocrit while chronic exposure of fish to pH>4.2 had no effect on blood hematocrit (see ref. Wood and McDonald, 1982). Chronic exposure was designated by Wood and McDonald (1982) as greater than 7 days and acute exposure 7 days or less. Fish that survive chronic exposure to low pH water appear to eventually compensate for hematological changes that occur during the acute exposure period. The increase in hematocrit associated with acid exposure is believed to be a result of at least three processes; erythrocyte swelling, a reduction in plasma volume, and mobilization of erythrocytes from the spleen (Milligan and Wood, 1982). Most studies monitoring acid-base balance, hematological disturbances, plasma ions and other . physiological parameters have been performed on fish exposed to pH values below pH 4.5 for acute-exposure periods. Plasma osmolarity decreased in fish exposed to high ambient Ca2+ concentration relative to fish maintained in low external calcium concentrations. This decrease may have been due to a drop in plasma ion concentrations. McDonald et al., (1980) found plasma Na+ and C1- concentrations were significantly lower in rainbow trout acclimated to high c a 2 + , low salt concentration, compared with fish acclimated to soft water. Their results make a good comparison since c~(NO,), was used to increase the water calcium concentration to approximately the same concentration (1.35 m ~ ) as in the present study. Wendelaar Bonga and Van Der Meij, (1980) also observed a significant decrease in plasma osmolarity in trout exposed to calcium-enriched (20.6 mEqeL-'1 freshwater. However, these authors did not observe any concurrent changes in plasma Na+, C1- or c a 2 + concentrations making the reason for the drop in plasma osmolarity obscure. Plasma N a + and C1- levels were not measured in the present study. Therefore the reason for a decrease in plasma osmolarity is not completely clear. However, since osmotic water inflow increased in the gills of fish adapted to calcium-enriched freshwater ( ~ i g .11, urine flow may have also been enhanced. This may have increased the renal loss of Na+ and C1- (Lock, 1979) accounting for the drop in plasma osmolarity. The reduction in plasma osmolarity may partially be a . result of changes in the transepithelial potential (TEP). Changes in pH and c a 2 + concentration of the external media can cause reversible changes in the TEP (Mc~illiamsand Potts, 1978). In their study, acidification of the external media caused the TEP to reverse from negative(inside) to positive(inside). Increasing the Ca2+ concentration also produced a reversible change in the TEP in a positive direction, since C1- diffuses out more rapidly than Na+ when the C a + 2 concentration exceeds 0.5 mM. Changes in the diffusional fluxes of these monovalent ions due to positive shifts in the TEP across the gills may account for loss of ions responsible for changes in plasma osmolarity. Although TEP was not measured in the present study the environmental calcium concentration in the calcium enriched groups was 1.5 mM, which would have been adequate to cause a positive shift in the TEP. Changes in water content between different tissues (kidney, liver, muscle) and the blood, may have accounted for the decrease in plasma osmolarity of trout exposed calcium-enriched freshwater. Oduleye (1975) found environmental calcium concentration affected total body water content in brown trout. This author also observed that body water is contained within different tissue compartments and these various tissues exchange water with the blood at different rates. Plasma Ca2+ concentrations and C a + 2 - ~ ~ ~ activity ase were not affected by exposure to pH 5.0 suggesting that calcium metabolism in immature trout or trout in the previtellogenic period is not affected. Therefore, disturbances in calcium metabolism caused by exposure to low pH, in trout in the process of vitellogenesis.(see Chapter II), are probably not a result of the inhibition of calcium transport at the gills. Whether active Ca2+ uptake in fish exposed to low pH is inhibited has not been previously studied. However, branchial ca2+ fluxes in rainbow trout exposed to low pH were measured by McDonald and Wood (1981) and did not change significantly. Other ATPase ion transport systems have been measured by Johnstone e t al., (1983) in the gills of Atlantic salmon (Salmo s a l a r ) exposed to low pH. These authors found no changes in ~ g ~ + - ~ T P aand s e Na+-ATPase enyzme activities in fish exposed to pH 4.9 relative to those in control fish. Inhibition of Mg2+-ATPase activities (~ohnstonee t al., 1983) and Na+ K+-ATPase activities (Johnstone e t al., 1983; Saunders e t al., 1983) did not occur in salmon until they were reared in freshwater acidified to pH 4.7 and below. In the present study a decrease in C a 2 + - ~ ~ p a s enzyme e activities were observed in fish exposed to calcium-enriched freshwater. When the external ca2+ concentration is lower than the blood ca2+ concentration a passive efflux of calcium occurs (~ilhaude t al., 1977). If the concentration gradient is decreased by increasing the ambient c a 2 + concentration then the passive loss of Ca2+ should be reduced. A reduction in Ca2+ . efflux as the external media increases in Ca2+ concentration might reduce the requirement for active transport of C a 2 + into the fish. These results are in agreement with some of the work reported in the literature but at variance with a few other studies. Fenwick ( 1 9 7 9 ) reported a higher ca2+-activated ATPase activity in the gills -of freshwater eels than in seawater acclimated eels. Low affinity Ca2+-ATPase activity increased in freshwater adapted Gillichthys in comparison to a hypersaline group (170%) although no differences were observed between seawater and freshwater adapted fish oneen en, 1981). Ma ( 1 9 7 6 ) also observed no changes in C a * + - ~ ~ P a activity se when fish were transferred from freshwater to seawater. Absolute C a 2 + - ~ ~ p a s e enzyme activity in roach (Rutilus rutilus L ) did not differ after exposure to high or low external Ca2+ concentrations for seven weeks (Shephard, 1981). However, field studies by Shephard ( 1 9 8 1 ) showed fish living in waters of varying calcium concentrations had enzyme activities proportional to the external calcium concentration. Umehara and Oguri ( 1 9 7 8 ) also found little change in the activities of Ca2+-A~Pasesin the gills of goldfish after 3 weeks exposure to calcium enriched freshwater. Only one study, by Burdick et al. ( 1 9 7 6 1 , reported higher ca2+-A~paseenzyme activities in the gills of killifish adapted to calcium-enriched freshwater in comparison with those fish adapted to freshwater. Higher Ca2+-ATPase activites in seawater adapted fish in comparison with freshwater adapted fish have been reported by some authors (~urdicket al., 1976; Ho and Chan, 1980). Differences in the literature regarding Ca2+-ATPase activities in the gills of fish exposed to different levels of environmental calcium, may be partially related to differences in the renal excretion of Ca2+ in different species, time of adaptation of the fish and differences in Ca2+ concentration of the assay media for assaying optimum activity of the enzyme. Shephard ( 1 9 8 1 ) suggests this enzyme may be capable of short-term responses to a change in external calcium concentration. Further studies of fish populations in hard and soft water, and chronic exposure to calcium-enriched water under laboratory controlled conditions, are required to determine the properties of this enzyme. Inhibition of Ca2+-ATPase enzyme activities in fish adapted to calcium-enriched freshwater may have been due to the release of an inhibitory factor 'hypocalcin' (pang et al., 1 9 7 4 ) . This compound is secreted from the Stannius Corpuscles (~urdicke t al., 1976; f en wick, 1976; Ma, 1976; So and Fenwick, 1 9 7 9 ) . Ca+2-AT~aseactivity increased when calculated on the basis of tissue weight in stanniectomized eels in comparison with sham operated eels (Fenwick, 1 9 7 6 ) . The activity of the Stannius Corpuscles is greater in seawater adapted killifish than those held in Ca2+ deficient seawater (pang, 1 9 7 3 ) . Doneen ( 1 9 8 1 ) suggested that Ca2+ transport in fish gills may involve both low and high affinity Ca2+-ATPases operating at different sites or by different mechanisms. Ca+2-ATPase assays . were measured for low ( 3 mM) affinity and high affinity ( 1 0 0 UM) activities in the present study. This would give an indication whether changes in environmental pH and/or Ca2+ concentrations affect these C a 2 + - ~ T p a ~ kinetic e forms differently. The activity of the low affinity ATPase decreased in fish exposed to the calcium-enriched freshwater, independent of pH, while the high affinity form did not change significantly. Doneen (1981) observed similar changes in Gi 1 l i c h t h y s where the low af f inity activity was higher in freshwater adapted fish compared with seawater (170%) adapted fish while the high affinity activity remained unchanged. Osmotic Water Uptake Fish exposed to low pH and low environmental calcium, displayed a tendency for an increased gill osmotic water influx (Fig. 14). This trend is likely caused by a loss of bound ca2+ from the surface of the gill membranes and a resultant increase in gill permeability ( ~ c ~ i l l i a m s1983). , Increased branchial osmotic water uptake may be balanced by an increased renal output as well as water entering intracellular compartments. Exposure of rainbow trout to pH 4.0 -4.5 has shown a net loss of Na+ and C1- across the gills resulting in electrolyte shifts from intracellular to extracellular compartments with water moving in the opposite direction (Wood and McDonald, 1982). Although plasma N a + and C1- were not measured in the present study, a reduction in plasma Na+ and C1- of fish exposed to low . pH has been observed in other studies ( ~ c ~ e o wetn a1 , 1984; McDonald & Wood, 1981; McWilliams & Potts, 1978, Spry et a l . , 1981). The increase in osmotic permeability of the gills in fish exposed to Ca2+-enriched freshwater at both pH levels is in contrast to other studies that show increasing the ambient C a 2 + concentration reduces the osmotic and ionic permeabilities of fish gills ( ~ o t t sand Fleming, 1970, 1971; Ogawa, 1974; Isaia and Masoni, 1976; Wendelaar Bonga and Van der Meij, 1981). This increase is possibly a result of the higher water osmolarity of the calcium-enriched groups. Wendelaar Bonga and Van der Meij (1981) found that the gills of fish exposed to a hypo-osmotic media ([Cab1=0.8 mM) showed an increase in the net osmotic water flow rate with increasing osmolarity of the ambient media, becoming maximal when isosmotic. Therefore, the osmotic permeability of the gills is inversely related to the magnitude of the osmotic gradient between the plasma and external media when the ambient media is hypo-osmotic (Wendelaar Bonga and Van der Meij, 1981). In media of high Ca2+ concentration (10 mM Ca2+ Wendelaar Bonga and Van der Meij (1981) found ambient osmolarity had no effect on osmotic water inflow which is always low. As the external media becomes more hyposmotic to the blood of fish, the gills become less permeable to water to reduce hydration. The increase in osmolarity between the low and high calcium groups may have had a greater effect on increasing gill permeability, than the increase in Ca2+ concentration to reduce gill permeability. In a hyposmotic media, high calcium concentrations (10 mM) have a greater effect on reducing gill permeability than osmolarity has on increasing it. The majority of studies cited above, showing that external calcium reduces the osmotic permeability of fish gills, have used calcium concentrations of 10 mM Ca2+. This may partially account for the discrepancy between the present study and other studies. However, Potts and Fleming ( 1 9 7 0 ) found ~ an increase in the exchange of tritiated water in the gills of F u n d u l u s when the ambient Ca+' concentration was reduced to 0.1 mM CaZ+ from 1.0 mM Ca2+. Whether differences in the osmolarities of the media used by Potts and Fleming, (1970) or species differences are the cause of the discrepancy between their study and the present study is not clear. An alternate explanation may be the difference in techniques, Potts and Fleming ( 1 9 7 0 ) measured the exchange of tritiated H 2 0 in the intact animal, in comparison with the measurment of osmotic water inflow of isolated gills used in the present study. McWilliams (1982) found that calcium had its greatest influence on gill permeability to ions at concentrations between 0 and 1 mM Ca2+ and increasing the ca2+ concentration above 1 mM produced no further effect. This suggests that the threshold level at which the ambient c a 2 + concentration affects gill permeability may be higher for osmotic compared with ionic regulation, since an external calcium concentration of 1.5 mM . Ca2+ did not reduce osmotic water inflow. Although ionic and osmotic regulation are often linked, Kostechi and Jones (1983) found a higher mortality in rainbow trout due to the effects of ionic stress in comparison to osmotic stress. In summary, the results of this study suggest calcium metabolism is not affected in trout exposed to intermediate acidic pH levels (pH 5.0-5.1). This is based on the observations that plasma calcium levels and gill C a 2 + - ~ ~ p a activities se did not change in trout after exposure to low pH. It is possible that pH 5.0 represents a threshold pH level and that increasing the H + concentration above this level may significantly affect the calcium balance in trout. E. Summary The results of this study show that pH levels 5.5 and less reduce the survival of kokanee and sockeye saimon eggs and alevins. Exposure of mature fish to pH 5.6 and below prior to ovulation and spawning also increases embryo mortality. Further, vitellogenesis in rainbow trout exposed to pH 5.0 was inhibited and may result in non-viable eggs or a failure of trout to spawn. The effects of low pH on egg quality and production in broodstock fish may be more important to the survival of fish populations than losses due to egg and alevin .mortalities. Studies only investigating the effects of low pH on embryo survival and not incorporating the effects on vitellogenesis and egg maturation are under-estimating lethal pH levels affecting the survival of fish populations. Reduced vitellogenin production in acid exposed fish may have been caused by the absence of an increased plasma calcium ion concentration. Low plasma calcium levels may be attributed to an inhibition of calcium uptake and/or an increased loss of calcium from the body. However, no effect of low pH exposure of se activity was observed. rainbow trout on gill C a 2 + - ~ ~ p a enzyme Although not measured, loss of calcium through the renal system and from skin and scales may have been significant enough in acid exposed fish to inhibit vitellogenesis, while not severely disturbing normal plasma calcium ion levels. Alternatively, an inhibition of the endocrine system controlling the synthesis and release of vitellogenin may have occurred in rainbow trout exposed to pH 5.0 accounting for both low plasma vitellogenin and calcium ion levels. Fish held in water enriched with calcium had higher plasma vitellogenin levels, suggesting environmental calcium concentration benefitted the reproductive the health of the fish. Gill Ca2+-ATPase activity and gill osmotic water inflow were also decreased and increased, respectively, in fish exposed to water enriched with calcium. Although no significant interactive effects of c a 2 + and pH were observed, the data did suggest an ameliorating influence of calcium on the physiological responses of salmonids to low pH exposure. REFERENCES Albro, F.W. (1975) Determination of protein in preparations of microsomes. Anal. Biochem. 64:485-493. Almer, B., W. Dickson, C. Ekstrom and E. Hornstrom (1974) Effects of acidification on Swedish lakes. ~ m b i o3: 30-36. American Public Health Association (APHA). (1975) Standard Methods for the examination of Water and Wastewater, 14th Edition. Prepared and published jointly by: American Public Health Association (APHA), American Water Works Association (AwWA), and Water Pollution Control Federation (WPCF) Joint Editorial Bd: Rand, M.C. (WPCF), Greenberg, A.E. (APHA), and Taras, M.J. (AWWA). M.A. Franson managing editor. 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