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Some effects of dehydroabietic acid (DHA) on hydromineral balance and other physiological, parameters… Kruzynski, George M. 1979

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SOME EFFECTS OF DEHYDROABIETIC ACID (DHA) ON HYDROMINERAL BALANCE AND OTHER PHYSIOLOGICAL PARAMETERS IN JUVENILE SOCKEYE SALMON ONCORHYNCHUS NERKA by GEORGE M. KRUZYNSKI B . S c , M.Sc, S i r George Williams University, 1968, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1979 (c) George M. Kruzynski, 1979 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag r ee tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f -Z<5C*tiC>&J The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date AJQi/ 6>/79 ABSTRACT Laboratory experiments were conducted to study the e f f e c t s of dehydro-a b i e t i c a c i d (DHA) on the physiology of the adaptation of sockeye salmon smolts (Oncorhynchus nerka) to sea water. Dehydroabietic acid occurs i n the r o s i n of commercially important coniferous trees and i s found i n the untreated e f f l u e n t s of the pulp and paper industry at concentrations acutely to x i c to salmonids. As t h i s r e s i n a c i d i s known to be one of the more p e r s i s t e n t t o x i c components of k r a f t m i l l e f f l u e n t (KME) and although i t s concentrations are greatly reduced by b i o l o g i c a l treatment, DHA i s nevertheless discharged i n the e f f l u e n t s of the pulp m i l l s s i t u a t e d on the Fraser River system as w e l l as o f those l o c a t e d on the coast of B r i t i s h Columbia. As sockeye salmon u t i l i z e both the Fraser and Thompson Rivers during t h e i r downstream migration, t h i s species may be exposed to DHA before entering the sea. An attempt was made to simulate t h i s s i t u a t i o n i n the laboratory by exposing sockeye salmon smolts to a sublethal concentration of DHA (0.65 mg/L) i n f r e s h water f o r 120 h and then t r a n s f e r r i n g them in t o sea water (28 °/oo) containing no DHA. Hydromineral balance was studied by monitoring changes i n plasma , . . , + + + + + + o s m o l a l i t y , plasma Na , K , Ca , Mg and C l , blood hematocrit and muscle water content a t the end of the freshwater DHA exposure and at 24 h i n t e r v a l s during the adaptation to sea water (120 h ) . A f t e r 24 h i n sea water the g i l l permeability to water and the water transport a b i l i t y of the gut were a l s o determined. Supportive experiments measured changes i n the s i z e of red blood c e l l s , the l e v e l s of plasma b i l i r u b i n as w e l l as the uptake and t i s s u e d i s t r i b u t i o n of DHA i n sockeye salmon smolts. L i p i d extracts of various t i s s u e s were analyzed f o r DHA residues by gas chromatography coupled with mass spectrometry (GC-MS). The exposure of sockeye salmon to DHA i n fresh water res u l t e d i n a hydromineral disturbance characterized by a drop i n plasma osmolality, sodium, and c h l o r i d e , i n d i c a t i n g a general hydration which was r e f l e c t e d by increased muscle water content. A lowering of d i s s o l v e d oxygen to 75% saturation markedly increased the t o x i c i t y of DHA and the osmotic imbalance may have been a secondary r e s u l t of an adaptive r e s p i r a t o r y response to a hypoxic stress brought on by DHA exposure. Increases i n blood hematocrit were caused by a swelling of the red blood c e l l s r e l a t e d to lowered plasma osmolality. When these f i s h were tr a n s f e r r e d to sea water, the hydration was replaced by dehydration and a r i s e i n osmolality was caused by abnormally elevated l e v e l s of a l l the plasma ions. The added s a l i n i t y s t r e s s r e s u l t e d i n some m o r t a l i t y and considerably greater excursions i n plasma e l e c t r o l y t e s occurred i n f i s h which were experiencing locomotor d i f f i c u l t y . Plasma . magnesium showed the greatest e l e v a t i o n and took the longest (96 h) to return to normal l e v e l s . P r i o r DHA exposure increased the permeability of the g i l l . During acute DHA exposure i n fresh water a gradual d e t e r i o r a t i o n i n schooling and f r i g h t response was followed by h y p e r s e n s i t i v i t y to mechanical s t i m u l i and abnormal swimming behavior. After sublethal exposure, the reduction i n schooling and f r i g h t response generally became most evident during the f i r s t 24 h of sea water adaptation. These r e s u l t s of the study are discussed i n terms of the p o s s i b l e r o l e s played by the g i l l s , gut and kidney i n the DHA-induced perturbations of hydromineral balance. The implications of the accompanying a l t e r a t i o n s i n behavior are discussed i n the context of the e c o l o g i c a l s u r v i v a l of sockeye salmon smolts during adaptation t o sea water. Residue analyses showed that sockeye salmon accumulated DHA from the water to high l e v e l s i n the b r a i n (954 x), l i v e r (428 x) and kidney (404 x) as w e l l as i n other t i s s u e s . The presence of DHA metabolites i n the b i l e , which a l s o contained the highest DHA residues (647.3 yg/g), i n d i c a t e s t h a t the h e p a t o b i l i a r y route i s important i n the excretion of DHA by f i s h . The p o s s i b i l i t y of the bioaccumulation of DHA by f i s h i n the w i l d i s discussed i n r e l a t i o n to the s e t t i n g of water q u a l i t y c r i t e r i a f o r pulp m i l l e f f l u e n t . i v TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 GENERAL MATERIALS AND METHODS 18 SYNOPSIS OF STUDIES ON DHA 31 PART I- PRELIMINARY EXPERIMENTS A. ACUTE TOXICITY OF DHA TO JUVENILE SOCKEYE SALMON INTRODUCTION 33 MATERIALS AND METHODS 34 RESULTS AND DISCUSSION • 37 B. EFFECTS OF ACUTE DHA EXPOSURE ON OSMOTIC BALANCE INTRODUCTION 43 MATERIALS AND METHODS 43 RESULTS AND DISCUSSION 46 PART I I PRINCIPAL EXPERIMENTS A. EFFECTS OF SUBLETHAL DHA EXPOSURE ON HYDROMINERAL BALANCE IN SOCKEYE SALMON SMOLTS INTRODUCTION 53 MATERIALS AND METHODS 54 RESULTS 55 B. EFFECTS OF DHA ON GILL AND GUT PERMEABILITY TO WATER INTRODUCTION .79 MATERIALS AND METHODS 80 RESULTS 87 DISCUSSION 91 PART I I I ECOLOGICAL IMPLICATIONS 113 APPENDICES 119 LITERATURE CITED 169 V LIST OF TABLES Table I II I I I IV V VI VII VIII IX XI XII XIII XIV XV XVI Chemical and p h y s i c a l c h a r a c t e r i s t i c s of well water used i n continuous flow bioassays with DHA. F i s h s i z e and acute bioassay operating parameters. Condition f a c t o r s of underyearling sockeye salmon exposed to acutely l e t h a l and sublethal concentrations of DHA i n f r e s h water (Expts. B and C). Condition f a c t o r and t o t a l body water i n underyearling sockeye salmon which died during exposure to 0.95 mg/L DHA i n f r e s h water (Expt. D). Percentage water of stomachs dissected from "swollen" and "normal" salmon exposed to 1.11 mg/L DHA (Expt. A). Muscle water i n underyearling sockeye salmon exposed to DHA i n fresh water and sampled at the d r i f t i n g stage. Muscle water i n underyearling chum salmon exposed to DHA i n sea water. Size of the sockeye salmon used i n the e l e c t r o l y t e balance experiments (Expts. 1, 2 and 3). Plasma i o n i c composition, hematocrit and muscle water content of sockeye salmon (Expts. 1, 2 and 3). Plasma e l e c t r o l y t e l e v e l s i n sockeye salmon during sea-water adaptation (Expts. 1, 2 and 3). Hematocrit of sockeye salmon (Expts. 1, 2 and 3). Percentage water i n gut of sockeye salmon (Expt. 2). Percentage water i n muscle of sockeye (Expts. 2 and 3). The plasma i o n i c composition of an "exposed" f i s h which l o s t e quilibrium a f t e r 72 h i n sea water (Expt. 1). Size, percentage muscle and gut water and symptoms i n f i s h which appeared to be se r i o u s l y a f f e c t e d during the course of Expt. 2. F i s h size and the loss i n weight during sea-water incubation of g i l l arches i s o l a t e d from j u v e n i l e sockeye salmon. Pages 20 35 47 48 49 50 52 56 59 63 65 67 69 71 72 89 V I Table XVII XVIII XIX XX XXI XXII XXIII XXIV XXV XXVI F i s h s i z e and the l o s s i n weight of i s o l a t e d i n t e s t i n a l sacs taken from ju v e n i l e sockeye salmon. The e f f e c t of f i s h loading density on concentration of DHA i n aquarium water. Comparison of concentrations of DHA recovered from f i l t e r e d and u n f i l t e r e d water samples. Concentrations of DHA d i r e c t l y soluble i n f r e s h water at pH 6.76 and 20°C. DHA residues i n the amphipod Anisogammarus con f e r v i c o l u s exposed to 0.4 mg/L DHA f o r 120 h i n sea water of s a l i n i t y 10 °/oo. Size, hematocrit and plasma i o n i c composition of f i s h deleted from data of Expt. 1. Size, hematocrit and plasma i o n i c composition of f i s h deleted from data of Expt. 2. Blood chemistry of sockeye salmon smolts exposed to 0.65 mg/L DHA f o r 5 days i n Expt. PB-1. Blood chemistry and s i z e of sockeye salmon smolts exposed to 0.65 mg/L DHA f o r 5 days i n Expt. PB-2. Red blood c e l l dimensions of ju v e n i l e sockeye salmon exposed to 0.65 mg/L DHA f o r 120 h i n fresh water. Pages 90 122 125 128 145 152 153 160 161 168 v i i LIST OF FIGURES Figure Pages 1 Map showing the l o c a t i o n of pulp m i l l s i n B r i t i s h Columbia and 2 the major sockeye salmon migration routes of the Fraser River system. 2 Seasonal presence of sockeye salmon i n the Fraser River i n the 4 region of the k r a f t pulp m i l l s at Prince George and Quesnel. 3 The seasonal presence of sockeye salmon i n the Thompson River 5 downstream from the k r a f t pulp m i l l at Kamloops. 4 Structures of some i s o l a t e d components of k r a f t m i l l waste 9 t o x i c to salmon. 5 Water supply system as used f o r continuing flow bioassays 19 with DHA. 6 I l l u s t r a t i o n of the arrangement of the donut tanks used f o r 22 continuous flow bioassays with DHA. 7 A section of two donut tanks showing d e t a i l s of the water 23 supply and the DHA metering system. 8 The cross-section of a donut tank g i v i n g d e t a i l s of the water 24 propulsion and waste trap systems. 9 T o x i c i t y curves i l l u s t r a t i n g 96 h LC50 values f or DHA to 39 ju v e n i l e sockeye salmon i n fr e s h water. 10 E f f e c t of reduced dissolved oxygen i n the water on the 41 t o x i c i t y of DHA to sockeye salmon smolts. 11 I l l u s t r a t i o n of the swelling of sockeye salmon caused by DHA 44 exposure. 12 Plasma e l e c t r o l y t e l e v e l s , hematocrit and muscle water content 60 i n sockeye salmon exposed to 0.65 mg/L DHA. 13 Change i n plasma e l e c t r o l y t e l e v e l s i n sockeye salmon during 62 sea-water adaptation following a 120 h exposure to 0.65 mg/L DHA i n fr e s h water. 14 Change of hematocrit and muscle water i n sockeye salmon during 66 sea-water adaptation. 15 Relations between muscle and gut water i n moribund f i s h 73 (Expt. 2). 16 Comparison of plasma e l e c t r o l y t e concentrations measured i n 3 75 d r i f t i n g f i s h (Expt. 2) I l l u s t r a t i o n of a section of a donut tank showing the p o s i t i o n i n g of p l a s t i c tubes to which sockeye salmon were confined. Detailed i l l u s t r a t i o n of one of the p l a s t i c tubes used f o r the containment of sockeye salmon. Di s s s e c t i o n procedure followed to make the g i l l preparation used i n the g i l l permeability experiment. Apparatus to measure weight l o s s of i s o l a t e d g i l l filaments. Diagram of apparatus used f o r incubation of i s o l a t e d i n t e s t i n a l sacs. E f f e c t s of f i s h loading density i n a s t a t i c bioassay on the acute t o x i c i t y of DHA to j u v e n i l e sockeye. D i s t r i b u t i o n of dehydroabietic a c i d i n pooled t i s s u e s of sockeye salmon smolts. Tissue d i s t r i b u t i o n of dehydroabietic a c i d in. a rainbow trout. Salmon m o r t a l i t y during the three e l e c t r o l y t e balance experiments (Expts. 1, 2 and 3). i x ACKNOWLEDGEMENTS I thank my supervisors, Dr. D.J. Randall f o r p r o v i d i n g the opportunity, and Dr. J.C. Davis f o r providing the f a c i l i t i e s f o r t h i s research. I am g r a t e f u l to both f o r t h e i r guidance and advice during r e v i s i o n s of the manuscript. S p e c i a l thanks are o f f e r e d to Dr. G. Greer f o r many f r u i t f u l discussions during the research and e s p e c i a l l y f o r h i s c o n s t r u c t i v e c r i t i c i s m during the preparation o f the t h e s i s . Thanks are also extended to Dr. I.H. Rogers f o r h i s advice and providing the use of GC-MS f a c i l i t i e s and to Mr. H. Mahood f o r advice and te c h n i c a l assistance during the residue analyses. The t e c h n i c a l assistance of Mrs. P. Futer, Mr. I. Shand and Miss B. Wishart i s also g r a t e f u l l y acknowledged as i s the cooperation o f the s t a f f at the P a c i f i c Environment I n s t i t u t e Laboratory, West Vancouver, B.C. where the research was conducted. This research was supported through F i s h e r i e s and Marine Service Science Subvention Program funding to Dr. D.J. Randall, a Quebec Government Post Graduate Scholarship to the author, and by in-house operating funds of the F i s h e r i e s and Marine Se r v i c e . A very s p e c i a l thanks to lay wife Jo-Anne who typed the manuscript, f o r her patience and constant encouragement through the d i f f i c u l t times. 1 GENERAL INTRODUCTION Wastes from the pulp and paper industry form the l a r g e s t s i n g l e source of i n d u s t r i a l e f f l u e n t being discharged i n t o r i v e r s and estuarine waters of B r i t i s h Columbia. At the same time, a l l f i v e species—^ of the anadromous P a c i f i c salmon must spend varying lengths of time i n r i v e r s and estuaries containing t h i s p o t e n t i a l l y t o x i c waste. Based on a v a i l a b l e information i t appears that i n the f i e l d t s a l m o n do not markedly avoid d i l u t e k r a f t m i l l waste and, i n f a c t , may sometimes be a t t r a c t e d to i t (Holland et a l . , 1960). In the Fraser River, the f r y of chum and pink salmon as well as y e a r l i n g chinook, coho and sockeye salmon are exposed to d i l u t e concentrations of k r a f t m i l l e f f l u e n t (KME) during the period of seaward migration. Sub-sequently, chum f r y and f i n g e r l i n g chinook and coho salmon feed i n estuarine areas f o r periods of up to several months (Williams et a l . , 1953; Dorcey et a l . , 1978); whereas sockeye salmon smolts may spend r e l a t i v e l y l i t t l e time i n brackish water before moving out towards the open sea (Williams, 1969). In the adult stage, a l l f i v e species of salmon must move through estuar-i e s where they spend v a r i a b l e lengths of time on t h e i r way to n a t a l streams. For example, the Adams River sockeye run delays approximately three weeks o f f the mouth of the Fraser River p r i o r to the freshwater t r a n s i t i o n . After entry into f r e s h water, some races of adult migrants spend up to three weeks i n the r i v e r before reaching t h e i r spawning grounds: e.g. Stuart and Bowron stocks (Figure 1) ( K i l l i c k , 1955). 1/ Oncorhynchus nerka sockeye O.kisutch coho O.keta chum O.gorbuscha pink O.tshawytscha chinook 2 Figure 1 . Map showing the l o c a t i o n of pulp m i l l s i n B r i t i s h Columbia and the major sockeye salmon migration routes of the Fraser River system. 3 During the period of r i v e r migration, whether as adults or smolts, these 5 3 f i s h must pass through waters r e c e i v i n g a t o t a l of 4x10 m per day of waste from the three pulp m i l l s at Prince George and one at Quesnel. The maximum concentrations of e f f l u e n t estimated to be present i n the Fraser mainstem i n the region of Prince George are i l l u s t r a t e d i n Figure 2 . During i t s passage through the Thompson River, the Adams River run, which provides 50-75% of the t o t a l commercial catch of Fraser sockeye (Gilhousen, 1960), would encounter the estimated e f f l u e n t concentrations shown i n Figure 3. Even though a l l the k r a f t m i l l s on the Fraser River system provide b i o -l o g i c a l (secondary) waste treatment, a study by Gordon and S e r v i z i (1974) showed that treated e f f l u e n t discharged at Prince George was acutely l e t h a l to salmon 90% of the time. Substandard treatment was also found at the Kamloops m i l l on the Thompson River ( S e r v i z i and Gordon, 1973). Some toxi c components of k r a f t m i l l waste are now known to survive b i o l o g i c a l treatment and i n s p i t e of continuing improvements i n these waste treatment systems, .... economic factors preclude the complete removal of these toxicants from the e f f l u e n t . Thus while the chronic dicharge of the more p e r s i s t e n t waste components i s l i k e l y to continue, l i t t l e i s known about t h e i r sublethal e f f e c t s on salmon i n the r i v e r . On the B.C. coast however, only 2 of the 11 m i l l s p r a c t i c e secondary treatment of effluent,consequently much higher l e v e l s of toxicants can be expected to reach estuarine or marine regions u t i l i z e d by salmon. Because of the extreme complexity and v a r i a b i l i t y i n the composition of whole k r a f t m i l l e f f l u e n t , the present study was r e s t r i c t e d to the consider-a t i o n of a s i n g l e component. Although t h i s n e c e s s a r i l y represents a gross s i m p l i f i c a t i o n of the possible b i o l o g i c a l e f f e c t s of whole KME i n the f i e l d , the component chosen (dehydroabietic acid-DHA) i s known to be one of the major t o x i c constituents found i n a wide v a r i e t y of wastes o r i g i n a t i n g from Figure 2-. The seasonal presence of sockeye salmon i n the Fraser River i n the region of the k r a f t pulp m i l l s at Prince George and Quesnel. The maximum t h e o r e t i c a l concentrations of e f f l u e n t to which these migrating salmon would be exposed , were c a l c u l a t e d on the basis of m i l l discharge r e l a t i v e to minimum mean monthly r i v e r volume flow'(1950-1970), assuming ra p i d and complete mixing. Figure 3; The seasonal presence of sockeye salmon i n the Thompson River downstream from the k r a f t pulp m i l l at Kamloops. The maximum t h e o r e t i c a l concentrations of e f f l u e n t to which these migrating salmon would be exposed were c a l c u l a t e d on the basi s of m i l l discharge as d i l u t e d by the minimum mean monthly r i v e r volume flow (1911- ' 1958), assuming rapid and complete mixing. 6 the f o r e s t products industry and i s c u r r e n t l y being discharged i n s i g n i f i c a n t amounts i n t o waters inhabited by migrating salmon. Brownlee e_t a_l. (1977) determined the h a l f - l i f e of DHA to be close to the 8 week value d e f i n i n g a " p e r s i s t e n t " organic compound (International J o i n t Commission, 1975) and stressed the need f o r chronic e f f e c t s studies with a view of e s t a b l i s h i n g safe l i m i t s . For the present study, a perspective of the problem researched can be gained by considering the c o n f l i c t of i n t e r e s t that e x i s t s i n the use of the Fraser River. In essence, t h i s r i v e r system remains one of the world's l a r g e s t producers of sockeye salmon but at the same time i t must assimilate a continuous input of i n d u s t r i a l wastes from f i v e pulp m i l l s . I t i s known that appreciable q u a n t i t i e s of DHA can survive the waste treatments p r a c t i c e d by these m i l l s , furthermore there continues to be a lack of information on the p o s s i b l e e f f e c t s of sublethal DHA exposure on salmon. Since sockeye salmon smolts must pass through these waters on t h e i r downstream migration, t h e i r a b i l i t y to cope with the normal st r e s s experienced during movement in t o s a l i n e waters may be a l t e r e d by exposure to pulp m i l l e f f l u e n t . Although we are ignorant of the a c t u a l behavior of salmon during t h i s f r e s h water (FW) to sea water (SW) t r a n s i t i o n i n the wild, laboratory experiments have shown that i t i s accompanied by changes i n water and e l e c t r o l y t e balance. Therefore the present study investigated the e f f e c t s of sublethal DHA exposure on the hydromineral balance of sockeye smolts i n fresh water and during adaptation to sea water. The experimental design was based on the premise that i f DHA i n t e r f e r e s with the mechanisms of osmotic and i o n i c homeostasis, the net r e s u l t w i l l be r e f l e c t e d i n a l t e r e d l e v e l s of the main plasma e l e c t r o l y t e s . Thus hydro-mineral balance was studied by measurement of plasma osmotic pressure and 7 plasma sodium, potassium, calcium, magnesium and chloride concentrations i n the blood of salmon during DHA exposure i n f r e s h water and a f t e r t r a n s f e r into sea water; muscle water content was measured as an i n d i c a t o r of t i s s u e water balance. In a d d i t i o n , the gut water transport a b i l i t y and the g i l l water permeability were determined as these organs are intimately involved i n the physiology of salmonid adaptation to sea water. F i n a l l y , observations were made on DHA-induced changes i n salmon behavior which may be of importance to s u r v i v a l during the t r a n s i t i o n from a freshwater to a marine existence. To keep the experimental design relevant to the natural s i t u a t i o n , attempts were made to simulate within the constraints of a laboratory study, several of the conditions that occur i n the w i l d . The f i s h were exposed to DHA while a c t i v e l y swimming f o r a period of time that the Adams River smolt migration i s estimated to take to reach the sea (5 days), and subsequent t r a n s f e r i n t o sea water was done at a rate thought to be representative of the t r a n s i t i o n from r i v e r to the sea during normal migration. Nature of the Toxicant The k r a f t process, i n essence, involves the a l k a l i n e d i g e s t i o n of wood chips which breaks down the l i g n i n and releases the f i b e r s . This i s follow-ed by bleaching, usually by c h l o r i n a t i o n , to obtain the desired brightness of the f i n a l pulp products. During t h i s process a number of natural products synthesized by the l i v i n g tree are s o l u b i l i z e d , extracted and washed out of the pulp. Common among these are the r e s i n acids and t h e i r d e r i v a t i v e s (alcohols, aldehydes and ketones-termed "unsaponifiables") which together with f a t t y acids occur i n the wood r o s i n of commercially important c o n i f e r s . Depending on t h e i r abundance, these products may be recovered as t a l l o i l soap. In the USA,resin a c i d from pine wood t a l l o i l forms the basis of an industry valued at $138 m i l l i o n ; i n Canada, where the lower 8 r o s i n content of pulpwood and high unsaponifiable y i e l d preclude economical recovery, r e s i n acids are usually discharged as wastes (Swan, 1973). Wash water from the bleaching process y i e l d s chlorinated l i g n i n d e r i v a t i v e s such as tetrachloroguaiacol and tetrachlorocatechol which are highly t o x i c to f i s h and whose s t r u c t u r a l s i m i l a r i t y to pentachlorophenol (Fig. 4), a w e l l -known uncoupler of oxidative phosphorylation, may suggest a s i m i l a r mode of toxic a c t i o n . S e r v i z i et a l . (1968) observed increased oxygen consumption i n sockeye salmon exposed to 0.1 mg/L of tetrachlorocatechol. Davis (1973) and Webb and Brett (1972) observed an increase i n oxygen requirements of salmon exposed to sublethal doses of k r a f t m i l l wastes. Food conversion e f f i c i e n c y was lowered and maintenance costs elevated by KME i n juvenile salmon i n studies by E l l i s (1967) and Webb and Brett (1972), while S e r v i z i et a l . (1966) ' observed a reduction i n the e f f i c i e n c y of yolk u t i l i z a t i o n i n alevins of sockeye and pink salmon exposed to 1% neut r a l i z e d bleach waste. However, as the composition of the mixed waste from a k r a f t pulp m i l l i s extremely v a r i a b l e and depends to a large extent on the wood species being pulped, the preci s e chemical state and i n t e r a c t i o n of these compounds i n re c e i v i n g waters remain l a r g e l y unknown. Although r e l i a b l e gas l i q u i d chromatographic (GLC) methods f o r i d e n t i f i -c a tion of r e s i n acids were developed 20 years ago (Hudy, 1959), they were not applied to environmental studies u n t i l 1968 by Maenpaa e_t al_. i n Finland, and s t i l l l a t e r i n North America (NCASI 1972, 1975; Rogers, 1973; Leach and Thakore, 1973). These techniques more recently combined with mass spectrometry (GC-MS), have confirmed the presence of DHA i n e f f l u e n t s from a v a r i e t y of f o r e s t products processes: s u l f i t e and k r a f t m i l l s (Maenpaa et a l . , 1968; Rogers, 1973; Leach and Thakore, 1973; Rogers et a l . , 1975), mechanical pulping (Row and Cook, 1971; Leach and Thakore, 1976), hardboard 9 DEHYDROABIETIC ACID LEVOPIMARIC ACID 1-ABIETIC ACID H0 oC CH, 2 \ ^ 3 H0 oC CH 0 NEOABIETIC ACID PALUSTRIC ACID OCH. TETRACHLOROGUAIACDL C l C l TETRACHLOROCATECHOL OH C l PENTACHLOROPHENOL Figure 4; Structures of some i s o l a t e d components of k r a f t m i l l waste t o x i c to salmon. Pentachlorophenol i s not found i n KME and i t s structure i s shown f o r purposes of comparison only. 10 plants (Row and Cook, 1971; Rogers ejt al_., 1977), and i n water from log storage areas (Fox, 1977). Only very recently have techniques been a v a i l a b l e to p r e c i s e l y quantify traces of r e s i n acids i n environmental samples. A k r a f t m i l l of average s i z e and p r a c t i c i n g secondary treatment can emit 50 kg of mixed r e s i n acids d a i l y because of the high(87 x 10 3 m3/day) e f f l u e n t discharge rates (Hrutfiord et a l . , 1975), and s t i l l meet the regulatory standards f o r t o x i c emissions. In s p i t e of such high discharge r a t e s , r e l a t i v e l y l i t t l e i s known about the subsequent b i o l o g i c a l fate of r e s i n acids i n the environment. However, Maenpaa et a l . (1968) found 0.17 mg/L r e s i n a c i d s i n lake waters at a distance of 4 miles from a k r a f t pulp m i l l i n F i n l a n d and the recovery o f r e s i n acids from the Fraser River 70 miles downstream from the three pulp m i l l s at Prince George (I.H. Rogers, personal communication) provides a d d i t i o n a l s i g n i f i c a n c e to the present i n v e s t i g a t i o n . The chemical reactions of r e s i n acids have formed a s i g n i f i c a n t p art of the larger study of terpenes i n c l a s s i c a l natural products chemistry. Some of the more important r e s i n acids such as p a l u s t r i c , levopimaric, and neoabietic (Fig. 4) are chemically unstable and e i t h e r oxidize or isomerize spontaneously to more stable forms such as a b i e t i c and dehydroabietic a c i d (DHA) during wood chip storage (Zinkel, 1975), or with heat and chemical treatment during the various pulping processes (Lawrence, 1959). The s t a b i l i t y of DHA can be a t t r i b u t e d to the presence of an aromatic r i n g which also can be predicted to make t h i s compound more water soluble than the other r e s i n a c i d s . Recent studies i n d i c a t e that DHA may be the most p e r s i s t e n t of the r e s i n acids a f t e r discharge i n t o the environment, both from natural and man-made sources. Simoneit (1977) reported DHA to be the most common r e s i n a c i d found i n l i p i d extracts of ocean sediment samples and suggested that DHA may 11 be an excellent natural b i o l o g i c a l marker of resinous higher plants for geochemical studies. Canadian studies i n a Lake Superior ecosystem have established DHA as being the most important p e r s i s t e n t organic contaminant 2 i n the sediment and water of a 25 km zone influenced by the discharge of a mixed-groundwood, k r a f t pulping p l a n t (Brownlee and Strachan, 1977; Fox, 1977). Brownlee et a l . (1977) estimate that 340 kg of DHA may be discharged d a i l y i n t o Nipigon Bay on Lake Superior. Disappearance due to b a c t e r i a l degradation appears slow and d i l u t i o n was suggested as the most s i g n i f i c a n t short-term removal mechanism (Fox, 1977). As r e s i n acids generally possess a low aqueous s o l u b i l i t y but are f r e e l y soluble i n f a t solvents, one would expect these compounds to pass r e a d i l y across the f i s h g i l l epithelium into the blood and thus become d i s t r i b u t e d throughout the body. Preliminary studies indicate that t h i s i n f a c t does happen both i n the laboratory and i n the f i e l d . In the Lake Superior study, rainbow t r o u t exposed to d i l u t i o n s of whole k r a f t m i l l e f f l u e n t s i n the laboratory accumulated DHA to a l e v e l 20 times that i n the water (Fox et a l . , 1977) and DHA was i s o l a t e d from native f i s h captured at a distance of 3 km from the m i l l discharge point (Brownlee and Strachan, 1977). The d i s t r i b u t i o n and the b i o l o g i c a l s i g n i f i c a n c e of DHA residues within the f i s h are unknown. The b i o l o g i c a l magnification of l i p i d soluble organochlorine i n s e c t i c i d e s i s now w e l l known and t h e i r a c t i o n i s often delayed u n t i l l i p i d reserves are u t i l i z e d . Such an a c t i o n could be of s i g n i f i c a n c e to migrating adult P a c i f i c salmon which cease feeding upon entry i n t o f r e s h water and r e l y on l i p i d reserves as a major energy source. Conceivably chlorinated l i g n i n d e r i v a t i v e s or r e s i n acids could follow s i m i l a r pathways i n f i s h . Storage of these toxicants i n c e l l u l a r l i p i d s may occur with r e s u l t i n g d i s r u p t i o n of c e l l u l a r function. Based on widespread 12 h i s t o l o g i c a l damage i n f i s h exposed to k r a f t m i l l waste i n f i e l d experiments (Fujiya, 1961; 1965) Warner and Tomiyama (in F u j i y a , 1965) suggested such a possible mode of action for r e s i n acids. An e a r l y clue to the p o s s i b l e fate of absorbed components of k r a f t m i l l waste was provided by Hagman (1936) who analyzed various organs of moribund f i s h c o l l e c t e d from a r i v e r below a k r a f t m i l l . Resin acids were found i n l i v e r , kidney, pancreas, and b r a i n t i s s u e , with highest l e v e l s i n the " l i q u i d which surrounds' the b r a i n i n the s k u l l c a v i t y " . Although the actual concentrations found are not given, the observation i s s i g n i f i c a n t . This author also described a v a r i e t y of biochemical changes which he a t t r i b u t e d to r e s i n a c i d buildup. The acute t o x i c i t y of mixed r e s i n acids to aquatic organisms has been known f o r many years. Hagman (1936) and Ebeling (1931) reported t o x i c i t y to f i s h as being i n the 1-2 mg/L range. More recent work has shown that mixed r e s i n acids are responsible f o r much of the r e s i d u a l t o x i c i t y i n e f f l u e n t s from the bleached k r a f t (Rogers, 1973), s u l f i t e (Maenpaa et a l . , 1968) and mechanical pulping processes (Leach and Thakore, 1976). Continuous-flow bioassays with p u r i f i e d i n d i v i d u a l r e s i n acids have established 96 h 2/ LC50's— f o r j u v e n i l e sockeye salmon to be l e s s than 1 mg/L (Rogers et a l . , 1975; G.M. Kruzynski, unpublished data). Based on acute t o x i c i t y alone, these compounds can be c l a s s i f i e d as "highly t o x i c " contaminants (Warner 1967; GESAMP, 1973). S a l i n i t y Stress A l l anadromous salmonids have the capacity of maintaining a r e l a t i v e l y constant blood osmotic pressure i n both f r e s h and s a l i n e waters, depending on 2/ 96 h LC50. Concentration l e t h a l to 50% of the f i s h i n 96 h. 13-the stage i n t h e i r l i f e c y c l e s . During migration and often w i t h i n a short time, a complete r e v e r s a l i n i o n i c and osmotic regulatory f u n c t i o n must take place both i n smolts moving i n t o sea water and i n adults entering f r e s h water. In f r e s h water, osmotic and i o n i c gradients are such th a t f i s h are faced with continual endosmosis of water and l o s s of s a l t s , as blood i s maintained hyperosmotic t o the surrounding water. As a r e s u l t of these unavoidable passive movements of e l e c t r o l y t e s , a salmon i n f r e s h water must a c t i v e l y absorb s a l t s , p r i m a r i l y Na + and C l from the water. Water i s continuously excreted i n large volumes v i a the kidneys as d i l u t e urine along with a s l i g h t l o s s of s a l t s . The r e n a l and b r a n c h i a l l o s s of Na + and C l i s compensated by a c t i v e ion uptake by the g i l l s as w e l l as i n the d i e t . In sea water, the i o n i c and osmotic gradients are reversed from the freshwater s i t u a t i o n and water i s l o s t across the g i l l s while Na + and C l d i f f u s e i n t o the blood down a concentration gradient. To compensate f o r the water l o s t , the f i s h swallows sea water which i s absorbed by the gut along with the s a l t which must be excreted by a process i n v o l v i n g the transport of Na + and C l by the g i l l s against a concentration gradient (active transport) (Maetz, 1971). The primary r o l e s of the kidney are the conservation of water and the e x c r e t i o n of the d i v a l e n t ions (Mg , S0 4, Ca ) which are absorbed by the gut. The anadromous l i f e c y c l e of salmon has n e c e s s i t a t e d the development of mechanisms to counter the passive movements of ions and water i n both media. In summary (Wood and Randall, 1973a) these comprise: a) A c t i v e transport mechanisms i n the g i l l s which can pump ions against t h e i r net d i f f u s i o n a l f l u x e s . b) E f f i c i e n t kidneys to eliminate osmotic gains i n f r e s h water and to l i m i t water l o s s i n sea water. c) I n t e s t i n a l mechanisms t o replace water l o s t to the hypertonic (SW) environment. 14 Thus a successful t r a n s i t i o n from one medium to the other requires a s e r i e s of fundamental modifications i n the p h y s i o l o g i c a l function of these organ systems. P r i o r to the completion of these adjustments, blood and t i s s u e water and e l e c t r o l y t e l e v e l s depart s i g n i f i c a n t l y from the steady state; these are then returned to close to p r e - t r a n s i t i o n values and are subsequently maintained by a v a r i e t y of regulative functions. The period of departure from steady state values together with the time span required to bring e l e c t r o l y t e and water l e v e l s to a new steady state has been termed the "adjustive phase" while the maintenance of the new steady state i s termed the "regulative phase" (Houston, 1959a). The adjustive phase has been shown to l a s t approximately 36 hours (h) i n coho salmon (Smith et a l ^ , 'i971; Conte et a l . , 1966) and chum f r y (Black, 1951; Houston, 1959b) and 60-100 h i n rainbow t r o u t Salmo g a i r d n e r i (Conte et^ a l . , 1966). A t l a n t i c salmon (Salmo  salar) smolts required 100 h to adjust plasma Na + back to normal l e v e l s (Koch and Evans, 1959) although plasma osmotic pressure was adjusted i n only 4 h (Parry, 1960). + + - ++ During these adjustive periods, plasma Na , K , C l and Mg concen-t r a t i o n s increase r a p i d l y due to s a l t i n f l u x and loss of plasma water across the g i l l s . Continued dehydration stimulates the ingestion of sea water, which leads to a further r i s e i n plasma i o n i c l e v e l s imposing an added load on the newly a c t i v a t e d branchial s a l t excretory mechanisms. F i n a l l y , the kidney which has had to sharply reduce i t s a c t i v i t y as an organ of water excretion and s a l t r e t e ntion begins to excrete the d i v a l e n t ions absorbed by the gut (Smith et a l . , 1971). These profound changes i n hydromineral co n t r o l as w e l l as the departure of both blood and t i s s u e i on l e v e l s from the steady state during the adaptive phase are not without consequence. Houston (1959b) found that the a c t i v i t y 15 and c r u i s i n g speed of chum salmon f r y dropped sharply upon transf e r into sea water. This reduction l a s t e d approximately 36 hours and was found to correspond to increases i n body chloride and decreases i n body water during the adjustive phase. When these l e v e l s returned to the c h a r a c t e r i s t i c sea water values, swimming performance returned to normal. The author interpreted these r e s u l t s as an i n h i b i t i o n of neuromuscular function caused by e l e c t r o l y t e imbalance during the adaptive phase. G i l l permeability has been described as a compromise between r e s p i r a t o r y and osmoregulatory needs (Steen and Kruysse, 1964; Randall et a l . , 1967). As the maintenance of a large r e s p i r a t o r y surface area for gas exchange also provides a large surface for passive osmotic and i o n i c movements, any factor which serves to increase the perfusion of blood through the re s p i r a t o r y lamellae can be expected to increase these passive movements, dist u r b i n g hydromineral balance. Wood and Randall (1973 a,b,c.) observed an increased Na + e f f l u x and water i n f l u x i n rainbow t r o u t during exercise i n f resh water, to compensate for the osmotic water gain, urine flows then increased. As trout respond to hypoxia by increasing v e n t i l a t i o n volume and probably the proportion of blood passing through the r e s p i r a t o r y lamellae (Randall et a l . , 1967; Randall, 1970), concurrent hydromineral a l t e r a t i o n s could therefore be expected as a r e s u l t of the response to hypoxic conditions. In a study of the e f f e c t s of handling and anesthesia on brook tro u t i n fr e s h water, decreases i n plasma and ti s s u e ion l e v e l s were the r e s u l t s of i n -completely compensated endosmosis and increased branchial and renal e l e c t r o l y t e e f f l u x derived from a primary response to vascular hypoxia (Houston et a l . , 1971). 16 K r a f t M i l l Waste and Hydromineral Balance Respiratory d i s t r e s s has been shown to be one of the most common manifestations of exposure to k r a f t pulp m i l l wastes i n salmonids (Alderdice and Brett, 1957; Schaumburg et a l M 1967; Walden et a l . , 1970). Davis (1973) has shown that salmon w i l l sharply increase v e n t i l a t i o n volume i n response to sub-lethal exposure to KME. As reduced a r t e r i a l oxygen was observed, the f i s h would probably attempt to increase lamellar flow to make up f o r the oxygen demand as blood i s usually maintained at 85-95% saturation (Randall, 1970). Such responses associated with enhancement of gas exchange w i l l lead to increase of branchial fluxes and r e s u l t i n a l t e r a t i o n s of water and e l e c t r o l y t e balance. Responses such as increased v e n t i l a t i o n volume can have serious t o x i c o l o g i c a l as well a s . p h y s i o l o g i c a l implications. Lloyd (1961) has suggested that the majority of the increase i n t o x i c i t y of poisons i n water of low dissolved oxygen i s caused by the increase i n the rate of r e s p i r a t o r y flow, with a consequent increase i n the rate at which t o x i c substances reach the g i l l epithelium. This a p p l i e s to any environmental or p h y s i o l o g i c a l f a c t o r which increases v e n t i l a t i o n volume; a d i r e c t response of the f i s h to oxygen d e f i c i e n c y . Changes i n g i l l permeability and kidney function can also occur as a r e s u l t of the d i r e c t e f f e c t of toxicant a c t i o n , e s p e c i a l l y i f the toxicant combines with membranes or accumulates to high concentrations. Such changes can lead to an increase i n the rate of passive s a l t and water movements across the branchial epithelium, or the f i s h may become d i u r e t i c and lose e l e c t r o l y t e s i n the urine. Subsequent departures of plasma and ti s s u e e l e c t r o l y t e l e v e l s from normal values would be governed by the d i r e c t i o n of osmotic and i o n i c gradients between body f l u i d s and the surrounding medium 17 once the homeostatic mechanisms were overwhelmed. Smith et_ a l . (1971) suggested that any major decrease i n the rate of ++ ++ . kidney f u n c t i o n could cause accumulation of d i v a l e n t ions Ca , Mg i n marine salmon, or water i n freshwater-adapted stages. The accumulation of d i v a l e n t ions can i n t e r f e r e with swimming performance (Houston 1959b); a function of c r i t i c a l importance to migrating salmon. Preliminary observations of Smith et_ a l . (1971) showed a drop i n urine production i n salmon upon exposure to lowered d i s s o l v e d oxygen c o n d i t i o n s . In view of the observed in t e r f e r e n c e of some k r a f t m i l l waste components with r e s p i r a t i o n i n salmonids, the maintenance of water balance may be a f f e c t e d i n a s i m i l a r way. To summarize, sockeye salmon can encounter pulp m i l l wastes during three c r i t i c a l phases of t h e i r l i f e c y c l e : smolt migration and entry i n t o sea water, entry of sea water-adapted adults i n t o f r e s h water, and subsequent upstream migration to the spawning grounds. The t r a n s i t i o n between hypertonic £ hypotonic media requires adjustment and r e g u l a t i o n by a v a r i e t y of osmo-regulatory systems. The i n t e r f e r e n c e with the optimum function of these systems could lead to hydromineral imbalance and thereby reduce the e f f i c i e n c y of such e c o l o g i c a l l y c r i t i c a l a c t i v i t i e s as swimming, feeding and predator evasion. Thompson (1945) i n d i c a t e d that adult migrant sockeye, delaying more than 12 days would f a i l to reach the spawning grounds. Thus f o r a migrating salmon, the net r e s u l t of a s u f f i c i e n t reduction i n scope f o r a c t i v i t y (Fry, 1971) by an i n d i s c r i m i n a t e s t r e s s such as pulp m i l l waste could be equivalent to o u t r i g h t m o r t a l i t y caused by acute t o x i c i t y i n the r i v e r (Brett, 1958). The experiments which follow were designed to inv e s t i g a t e some e f f e c t s of DHA, a major tox i c component of pulp m i l l waste, on the hydromineral balance and related physiology of sockeye salmon smolts. 18 GENERAL MATERIALS AND METHODS Water Supply Experiments were conducted in the laboratories of the Pacific Environment Institute (PEI), West Vancouver, B.C., from 1973-1978. Fresh water supplied to the laboratory from a well was processed as shown in Fig. 5. Water passed through a cartridge f i l t e r (Cuno-Micro Kleen II, 125 um) and then through a bank of aspirators into a series of constant level foam-insulated header tanks equipped with refrigeration units (Frigid Units, Toledo, Ohio) where i t was vigorously aerated and chilled. Gas supersaturation was eliminated by means of a stripping column i n which a counter-current of oil-free a i r passed through a bed of glass marbles. Air-stones were used to bring the water supply to saturation. The chemical composition of the well water during the study period i s shown in Table I. The laboratory sea water supply i s drawn from a depth of 18 m in Burrard Inlet and was also f i l t e r e d , chilled and aerated before use. Sea water varied in salinity from 26-29 °/oo (^795-o 890 mOsM/kg) had a pH range of 7.6-7.9 and a temperature range of 11.5 ±0.5 C. The system illustrated in Fig. 5 supplied f i l t e r e d , a i r equilibrated water to the experimental tanks at constant temperature. The bottom header tanks provided each annular tank (donut) with SW and FW through separate lines and flowmeters (Manostat Predictability Flowmeter, 36-541-31) so that a change from one water supply to the other could be done separately at each tank by closing one flowmeter and opening the other. Thus "transfer" experiments were conducted without disturbance of the fi s h and at the water flow rates used, (500 mL/min) 95% replacement of FW by SW was accomplished within ^ 5.8 h (Sprague, 1969). 19 1 WELL WATER 1 VALVE 2 CARTRIDGE FILTER 3 ASPIRATORS 4 HEADER TANK 5 1 HP REFRIGERATION UNIT 6 OVERFLOW 7 GAS STRIPPING COLUMN 8 AIR SUPPLY TO SINTERED GLASS FUNNEL 9 AIR SUPPLY TO AIRrSTONE 1 0 V3 HP REFRIGERATION UNIT 11 WATER SUPPLY TO FLOWMETERS o SEA WATER t \ I™ 1 J / < J S — ' J&N 11 TO FLOWMET 6 ER < Figure 5 . Water supply system as used f o r continuing flow bioassays with DHA. Table I. Chemical and p h y s i c a l c h a r a c t e r i s t i c s of well water used i n continuous flow bioassays with DHA. Mean (Range) mEq/L Na + 0.79 (0.52-1.26) K + 0.04 (0.02-0.05) C a + + 0.78 (0.56-1.25) Mg + + 0.22 (0.13-0.34) C l " 1.00 (0.71-1.71) Hardness mg/L CaCO^ A l k a l i n i t y mg/L CaCC>3 Conductivity umho/cm pH^ " range o Temperature C Range of pH i n experimental tank water mg/L 18.2 (12.0-29.0) 1.43 (0.81-2.10) 15.6 (11.3-25.0) 2.68 (1.60-4.11) 35.4 (25.0-60.7) 49.2 (41.0-60.1) 27.4 (26.6-28.0) 218.2 (163-302) 6.86-6.96 11.5 ±0.5 6.71-6.96 21 The Annular Bioassay Tank System A l l experiments were conducted using a continuous-flow system. A s e r i e s of 12 annular f i b e r g l a s s tanks was supported on a two-tiered wooden frame i n a U-shaped array (Fig. 6) and supplied with water as shown i n F i g . 7. The tanks were a modif i c a t i o n of the "annular growth chambers" described by Kruzynski (1972). The mold was based on a r a d i a l t i r e inner tube (50 cm i d , 90 cm od) and was manufactured to s p e c i f i c a t i o n s by E v e r l a s t P l a s t i c s , North Vancouver, B.C. Urethane foam was sprayed on the outside f o r i n s u l a t i o n and the tank stood on wooden blocks as i l l u s t r a t e d i n F i g . 8, which also shows cr o s s - s e c t i o n a l view of the dr a i n area. De t a i l s of a tank module are shown i n composite form i n F i g . 7. The toxicant metering system i s i l l u s t r a t e d at the top and consisted of a 25 L glass Mariotte b o t t l e d e l i v e r i n g a concentrated stock s o l u t i o n to the tank through Teflon tubing (1.5 mm i d , 0.4 mm wall) supported by glas s tubing held i n a b i u r e t clamp (Leduc, 1966). A t o x i c a n t flow of 3 mL/min was mixed with a well water flow of 500 mL/min. The bottom of F i g . 7 i l l u s t r a t e s the r e -c i r c u l a t i n g pump connection to the stand pipe/water drive u n i t , the waste trap tubing i n the waste c o l l e c t i n g p o s i t i o n , and f i s h schooling under the dark-ened screen p o r t i o n ( r i g h t ) . A water pump (March MDX-3) r e c i r c u l a t e d the water at ^  25L/min and provided a water current around the tank with a v e l o c i t y determined by the stand pipe/water propulsion u n i t used. For experi-ments i n which f i s h were free swimming, the stand pipe u n i t shown i n F i g . 8b provided a current of ^ 15 cm/sec with the 70 L tank f i l l e d to a volume of 58L. In the g i l l permeability experiments (p.83) i n which f i s h were r e s t r i c t e d to tubes placed within the tank, a modified u n i t i l l u s t r a t e d i n F i g . 8c was used to maintain adequate water movement against the added r e s i s t a n c e of the tubes. Figure 6. I l l u s t r a t i o n of the arrangement of the donut tanks used for continuous flow bioassays with DHA. 23 . ^ From water supply 0 10cm Figure 7"". A section of two donut tanks showing d e t a i l s of the water supply and the DHA metering system. 25 The r e c i r c u l a t i n g pump was suspended from a nylon cord across the i n t e r n a l diameter of the tank to eliminate v i b r a t i o n . Each tank was equipped with an air-stone to maintain d i s s o l v e d oxygen l e v e l s above 90% saturation a f t e r i t appeared that the toxicant increased consumption i n exposed f i s h . Covers made of f i b e r g l a s s mosquito screening kept f i s h from jumping out, while a piece o f black p l a s t i c provided a shaded area. Salmon maintained p o s i t i o n against the current under t h i s covered area and then schooled t i g h t l y when disturbed. At feeding, p e l l e t s of OMP (Oregon Moist P e l l e t ) were dropped j u s t downstream from the stand pipe o u t l e t and were qui c k l y consumed by the f i s h as the p e l l e t s were c a r r i e d around the tank by the current. The tank incorporated a waste trap (Fig. 8a) which c o l l e c t e d un-eaten food and feces d r i f t i n g around the i n t e r i o r circumference of the tank wa l l . The trap was drained by a length of Tygon tubing which was kept a t water l e v e l f o r c o l l e c t i o n (Fig. 7) and lowered to the drain to siphon the accumulated waste. This was done immediately before the d a i l y morning feed-ing to drain waste accumulated overnight and about f i v e minutes a f t e r feed-ing to remove remaining food. Adjacent tank modules were separated by black p l a s t i c c urtains to prevent v i s u a l disturbance of the f i s h . Each module was equipped with a 20W fluorescent l i g h t (Duro Test V i t a Lite) providing an i l l u m i n a t i o n of 30-50 l x at the water surface. Natural photoperiod was maintained by a l i g h t sensor mounted on the roof of the laboratory b u i l d i n g . An incan-descent l i g h t bulb was l e f t on 24 h a day to provide a dim l i g h t simulating night-time outdoor l i g h t i n g conditions and to f a c i l i t a t e observations. No metal except f o r s t a i n l e s s s t e e l came i n t o contact with the water supply. Header tanks were of polyethylene (Nalgene), p i p i n g was of PVC and a l l tubing d e l i v e r i n g water to the glass flow meters was Tygon. The r e f r i g e r a t i o n u n i t s had Teflon coated components. The magnetic drive pump housings were polypropylene and latex rubber tubing supplied water to the PVC stand pipe u n i t s . The tanks were epoxy reinf o r c e d f i b e r g l a s s while the toxicant was delivered from glass b o t t l e s through Teflon tubing. F i s h Holding Sockeye salmon stocks were kept i n the outdoor f i s h - h o l d i n g f a c i l i t y at the P a c i f i c Environment I n s t i t u t e . As f i s h were obtained from a v a r i e t y of sources and at various stages of development, d e t a i l s are given i n the Materials and Methods section of each experiment. The outdoor holding tanks were of f i b e r g l a s s construction, measuring 3.1 x 1.2 m and were f i l l e d to a volume of 4000 L with well water whose chemical c h a r a c t e r i s t i c s have been described i n Table I. Water temperatures ranged seasonally from 10-12°C and the f i s h were fed twice d a i l y on a d i e t of Oregon Moist P e l l e t s . F i s h Transfer F i s h were dip-netted from the outside holding tanks i n groups of ^  20 and transferred i n t o a polyethylene bucket containing 33 mg/L MS-222 o (Tricaine methanesulfonate, Sandoz) dissolved i n water of s a l i n i t y 10 /oo at 11°C. F i s h were then placed i n t o the laboratory donut tanks where they were held f or a minimum of 96 h for a c c l i m a t i z a t i o n to the flowing water conditions i n the laboratory p r i o r to the s t a r t of an experiment. As the anesthesia was very l i g h t , f i s h were swimming normally and schooling under cover within 5 min and would feed within 1 h of introduction i n t o the tank. Feeding was done once d a i l y with OMP and was discontinued 24 h p r i o r to the s t a r t of experiments. Anesthesia and Blood Sampling F i s h were anesthetized i n two stages. A l i g h t anesthesia was brought on by the addition of 2g of MS-222 to the tank a f t e r shutting o f f the water 27 supply and adding an air-stone l i g h t l y bubbling i n t o the water. The anesthetic was dissolved i n IL SW and then slowly added to the tank where thorough mixing was ensured by the r e c i r c u l a t i n g pump. As soon as the f i s h began to d r i f t with the current, two f i s h at a time were dip-netted i n t o a polyethylene bucket containing 200 mg/L MS-222 for r apid terminal anesthesia. This s o l u t i o n was prepared by the addition of 1.2 g MS-222 to an oxygenated mixture of 3L SW and 4L FW, y i e l d i n g a s a l i n i t y of 10-12 °/oo at a pH ^6.3. This method ensured rapid anesthesia, minimized hypoxia and was meant to minimize the osmoregulatory stress involved i n un-buffered MS-222 anesthesia (Wedemeyer, 1970) and the p h y s i o l o g i c a l stress caused by the handling of f i s h i n so f t water (Wedemeyer, 1972). Aft e r immobilization, the f i s h were rinsed with deionized water, measured to the nearest mm, b l o t t e d and weighed to the nearest 10 mg on a Mettler P1200 or to 100 mg on a PS1200 top loading balance. The caudal peduncle was severed and blood was c o l l e c t e d i n t o Natelson heparinized c a p i l l a r y tubes. The tubes contained 6 USP ammonium heparin and were c h i l l e d on i c e p r i o r to blood c o l l e c t i o n . Depending on the s i z e of each f i s h , from 200-500 uL of blood was c o l l e c t e d and the tubes were sealed with Critocaps (Sherwood Medical Industries) and stored on i c e u n t i l a l l the f i s h had been processed. The tubes were placed i n c h i l l e d balsa-wood l i n e r s to minimize warming and centrifugated at 1300G f o r 20 minutes i n a DAMON/IEC Model CS centrifuge. At other times, blood was c o l l e c t e d i n microhematocrit tubes and centrifugated i n an IEC microhematocrit centrifuge Model MB f o r 3 minutes at 13,000G. The hematocrit was recorded, plasma was separated, transferred to 2 mL disposable c o n i c a l polystyrene sample cups (Technicon Auto Analyzer) and analyzed immediately or stored frozen. In cases where the water content of the f i s h was determined, each carcass was b l o t t e d dry with an absorbent wiper, wet-weighed i n a tared aluminum dish and dried i n a forced a i r oven at 110°C to constant weight. In cases where the "muscle" water content was determined, a cross-section of the f i s h was used. A f t e r the caudal peduncle had been transected and the blood sampling completed, a second transverse cut was made immediately p o s t e r i o r to the vent. This section consisted p r i m a r i l y of muscle but included the anal and adipose f i n , a section of the ve r t e b r a l column as well as the skin and scales. This preparation was then d r i e d as described above. The % water c a l c u l a t e d was termed "muscle" water. Blood E l e c t r o l y t e Determination Plasma chloride was measured on a Buchler-Cotlove Di r e c t Reading Chloridometer (Buchler Instruments D i v i s i o n , Nuclear- Chicago, N.J.) adapted for 10 uL samples with a rheostat provided by the manufacturer. A va r i a b l e volume Buchler Micropipet f i t t e d with a short length of PE-90 tubing was used to t r a n s f e r the plasma sample to the instrument and de-ionized water was used as the wash-out solvent. The Chloridometer was ca l i b r a t e d using NaCl solutions following manufacturer's i n s t r u c t i o n s and read d i r e c t l y i n mEq/L C l . Plasma osmolality was determined on a 1:1 d i l u t i o n with deionized water. Using an Eppendorf Micropipet, a 100 uL a l i q u o t of plasma was transferred to an osmometer v i a l followed by 100 uL deionized water using the same pipett e t i p . An Osmette-S-Semi-Automatic Osmometer (Precision Systems Inc., Mass.) c a l i b r a t e d with manufacturer's standards and operated i n the small-sample (200 uL) mode, was used to determine plasma osmolality (milliosmol (mOsm)/kg water). A f t e r a determination, the sample was thawed and 100 uL of the mixture was transferred with an Eppendorf Micropipet to a 4 ml conical sample cup containing 2 mL 0.25% strontium chloride (SrCl2> dispensed with an Oxford Pipettor Model R, then capped and mixed on a vortex stir r e r prior to cation analysis by Atomic Absorption Spectrophotometry (AAS). In cases where there was not enough plasma for osmometry, 50 yL of undiluted plasma was added directly to the SrCl^ solution. Strontium chloride was used according to the method of Paschen and Fuchs (1971) for suppression of anionic interferences during plasma analysis by AAS. A single dilution sufficed for analysis of the four + + ++ ++ cations Na , K , Ca , Mg Plasma cations were analyzed on a Perkin Elmer Atomic Absorption Spectrophotometer Model 403, u t i l i z i n g an air/acetylene flame. Na+, K+, Ca , Mg were analyzed at wavelengths (nm) 330 UV, 385 VIS., 211 VIS., and 285 UV respectively. A standard solution containing (in mEq/L) 140 Na+, 5.0 K+, 5.0 Ca + +, and 1.97 Mg + + was prepared according to methods described in the instrument manual and then diluted in a fashion identical to the unknown samples. Absorbance was read and the concentration of the various ions was calculated and expressed in mEq/L. Periodic checks of instrument performance were made using Hyland I and II and Dade Lab-trol and Patho-trol Chemistry Control Sera. Preparation of DHA for Fish Bioassays Dehydroabietic acid (DHA) was prepared by the method of Halbrook and Lawrence (1966) to a purity of 95.7% as determined by gas liquid chromatography (GLC). A concentrated stock solution was made by dissolving the required amount of DHA in 100 mL ethyl alcohol, adding 2.5 mL 5N NaOH followed by 100 mL d i s t i l l e d water. Light st i r r i n g with a magnetic s t i r bar and slow addition of the water ensured complete s o l u t i o n . The mixture was then slowly added to the Mariotte b o t t l e containing approximately 20 L d i s t i l l e d water and 2.5 mL 5N NaOH, again s t i r r i n g continuously. This concentrated stock s o l u t i o n 100-200 mg/L) was then made up to 25 L with d i s t i l l e d water. The b o t t l e was then connected to a water-operated vacuum pump (aspirator) f o r 10-15 min. This evacuation procedure combined with vigorous s t i r r i n g e f f e c t i v e l y de-gassed the solut i o n . As the Mariotte b o t t l e system operates under p a r t i a l vacuum, t h i s procedure eliminated subsequent problems a r i s i n g from the formation o f microbubbles which would coalesce i n the f i n e bore of the toxicant d e l i v e r y tubing di s r u p t i n g the flow. Using t h i s degassing procedure, the toxicant flows when once established, required l i t t l e or no adjustment. The b o t t l e was placed on a board at the center of the donut (Fig. 7) and at a flow rate of 3 mL/min provided toxicant f o r the duration of the 120 h exposure period with no further disturbance to the f i s h . In preliminary experiments, measurements (by GLC) of the concentrations of DHA a c t u a l l y present i n the water showed that ^ 90% of the t h e o r e t i c a l dosage had been attained (Appendix 1). 31 SYNOPSIS OF STUDIES ON DHA During preliminary experiments to e s t a b l i s h the acute t o x i c i t y of DHA to sockeye salmon i t became necessary to develop chemical methods to determine the actual concentrations of DHA present i n the water. These methods (the extraction of DHA from the water and i t s q u a n t i f i c a t i o n by g a s - l i q u i d -chroma tography (GLC)) were used to r e f i n e DHA s o l u b i l i z a t i o n techniques and subsequently to monitor DHA concentrations i n flow-through bioassays. When i t became confirmed that f i s h were removing DHA from the water during b i o -assays, extraction techniques were also developed to measure DHA residues i n f i s h t i s s u e and f i n a l l y i n f i s h food organisms. In a l l , f i v e experiments were done and the main fi n d i n g s are summarized below; the d e t a i l s are given i n Appendix I. The s u r v i v a l of f i s h i n what should have been an acutely t o x i c con-centration of DHA during a s t a t i c bioassay was due to a rapid reduction of the actual amount of DHA present i n the water (Appendix 1-1). Although t h i s was l a r g e l y a t t r i b u t a b l e to the presence of f i s h , some adsorption onto the walls of the t e s t aquarium was also indicated. On the basis of these r e s u l t s , a l l subsequent experiments were conducted under continuous flow conditions exceeding the toxicant/water replacement guidelines given i n Sprague (1969). In addition, a check of actual DHA concentrations present i n the water during these bioassays showed that 90-95% of the t h e o r e t i c a l dose was maintained during the duration of the exposure period. When DHA was prepared and mixed with w e l l water i n the form used for continuous-flow bioassays (as the sodium s a l t ) , f i l t r a t i o n had no e f f e c t on i t s recovery from the water (Appendix I~2). This experiment showed that the r e s i n a c i d was i n sol u t i o n , and as such should be av a i l a b l e to the f i s h . 32 Another experiment (Appendix 1-3) determined the d i r e c t aqueous s o l u b i l i t y of DHA to be 3.3 mg/L, i n d i c a t i n g that the free a c i d can dissolv e d i r e c t l y i n the water to concentrations exceeding those found to be acutely t o x i c to salmonids. As preliminary studies had shown that DHA was taken up by salmon during sublethal exposure i n fresh water, an experiment was conducted to determine whether accumulation i n the body d i d occur and i f so, to determine the ti s s u e d i s t r i b u t i o n of the toxicant (Appendix 1-4). The r e s u l t s confirmed that DHA was taken up by f i s h and accumulated to a l e v e l 30 times higher than that a v a i l a b l e i n the water. Much higher bioconcentration was measured i n i n d i v i d u a l organs such as the brain (954 x), kidney (428 x), l i v e r (404 x); the b i l e contained the highest o v e r a l l concentration of DHA (996 x). Gas chromatography coupled with mass spectrometry (GC-MS) was used to detect several metabolic d e r i v a t i v e s of the parent DHA molecule i n the b i l e , i n d i c a t -ing that the hep a t o b i l i a r y route i s involved i n DHA excretion i n sockeye salmon. The exposure of a representative f i s h food organism (the amphipod Anisogammarus confervicolus) to DHA resu l t e d i n a bioconcentration of 21 x that present i n the water (Appendix 1-5). These r e s u l t s i n d i c a t e that salmon may accumulate DHA through the food chain as w e l l as d i r e c t l y from the water. The Discussion w i l l go in t o the b i o l o g i c a l s i g n i f i c a n c e of these high DHA residues i n r e l a t i o n to feeding behavior and p h y s i o l o g i c a l function of the salmon. This concludes the summary of studies which were done on the toxicant and the next section w i l l cover experiments which investigated the d i r e c t e f f e c t s of DHA exposure on sockeye salmon. PART I. PRELIMINARY EXPERIMENTS A. ACUTE TOXICITY OF DHA TO JUVENILE SOCKEYE SALMON INTRODUCTION The acute t o x i c i t y of DHA to sockeye salmon was determined on three separate occasions under continuous-flow conditions with the purpose of est a b l i s h i n g the 96 h LC50 i n f r e s h water. The t o x i c i t y curves thus generated were then used to estimate a concentration of DHA which would cause n e g l i g i b l e m o rtality during the subsequent sublethal e l e c t r o l y t e balance experiments. Of the three acute bioassays, the f i r s t i n March 1974 (Expt. 74) u t i l i z e d a broad range of 6 concentrations (0.47 to 3.13 mg/L DHA) while the second i n March 1976 (Expt. 76) and t h i r d i n A p r i l 1977 (Expt. 77) employed a more r e s t r i c t e d range of concentrations which was expected to bracket the 96 h LC50. During the course of acute bioassays, salmon appeared to be under a res p i r a t o r y s t r e s s , as manifested by frequent coughing and v e n t i l a t o r y changes. Subsequent work showed that an elevation of hematocrit occurred during sublethal exposure to DHA, a response that i s known to occur during hypoxia (Doudoroff and Shumway, 1970). I f DHA was i n t e r f e r i n g with normal gas exchange or with the transport of oxygen by the blood, then a lowering of dissolved oxygen l e v e l s i n the water could increase the t o x i c i t y of the r e s i n acid. To t e s t i f t h i s was the case, an experiment was conducted on two groups of salmon smolts exposed to a normally sublethal exposure to DHA. In one group, dissolved oxygen (D.O.) l e v e l s were maintained at 'v* 75% saturation, while i n the other, dissolved oxygen was maintained ^ 90% saturation. Both groups of f i s h were exposed to a normally sublethal concentration of DHA (0.65 mg/L) f o r 120 h i n fresh water. MATERIALS AND METHODS The sockeye salmon used i n a l l three acute bioassays were of the Cultus Lake stock. Expt. 74 was conducted with f i s h which had been ra i s e d at PEI from eggs obtained from Cultus Lake i n November 1973. The 1976 f i s h were obtained as smolts i n January 1976 and had been kept at PEI for 2 months p r i o r to the experiment, while the 1977 f i s h were obtained as year-l i n g s and had been at PEI f o r 5 months p r i o r to use. At the time of the acute bioassays a l l f i s h were 17-18 months o l d and had the s i l v e r y c o l o r a t i o n c h a r a c t e r i s t i c of sockeye smolts. A f t e r t r a n s f e r from outdoor holding tanks according to procedures described i n General Methods, the f i s h were given 48 h to acclimatize to the laboratory tanks before the toxicant exposure was started. In Expt. 74, the f i s h had been i n the laboratory tanks for 2 months p r i o r to the bioassay. Feeding was discontinued 48 h p r i o r to the s t a r t of the exposure and f i s h were not fed during the experiments. Test conditions and f i s h s i z e are given i n Table I I . One co n t r o l tank was used i n each experiment and received the solvent c a r r i e r at the same rate as the t e s t tanks but no DHA. Continuous observations were made during the day while at night, m o r t a l i t i e s were recorded at approximately 4 h i n t e r v a l s . Death was judged by the absence of a l l movement upon handling. The time to death was recorded, the f i s h was measured to the nearest mm (fork length), b l o t t e d and weighed to the nearest 10 mg on a top-loading balance. Log p r o b i t paper was used to p l o t cumulative % m o r t a l i t y against time and the TL50 (time to 50% mortality) f o r each concentration was determined g r a p h i c a l l y ( L i t c h f i e l d , 1949). Acute t o x i c i t y curves were then p l o t t e d using TL50's vs concentration on lo g - l o g paper and the 96 h LC50's were Table I I . F i s h s i z e and acute bioassay operating parameters. Experiment 74 Fish Size Mean ±SE(n) Fork length Wet weight cm g 11.7 +0.06(141)1 17. 74 ±0.26 76 77 12.0 ±0.15( 44) 13.0 ±0.15( 59)127.06 +0.93 17.97 ±0.52 Water Characteristics (range) Number 20 12 15 Loading density per tank L/g fish/day Temperature °C pH 2.03 3. 34 1. 79 11.2 ±0.3 6.32 - 6.40 Dissolved oxygen % 2. 70-8S 10.5 ±0.5 7.04 - 7.13 90-95 11.2 ±0.5 6.80 " 6.B7 90-95 DHA mg/L , Concentrations Estimate of tested 3.13, 1.92, 1.65, 1.16, 0.61, 0.47 1.00, 0.87, 0.65 1.29, 1.00, 0.79, 0.63 96h LC50 0.50 0.79 0. 88 1 By i n t e r p o l a t i o n from t o x i c i t y curve. d i s s o l v e d oxygen dropped to 70% during the f i r s t 24 h of exposure and was returned by supplemental aeration to >85% f o r the remaining 96 h. Cn 36 estimated g r a p h i c a l l y by i n t e r p o l a t i o n from the t o x i c i t y curves. The absence of p a r t i a l m o r t a l i t i e s at 96 h precluded the c a l c u l a t i o n of a more precise LC50 estimate and confidence l i m i t s as recommended by Sprague (1969). In Expt. 74 the toxicant exposure period extended to 170 h while i n Expts. 76 and 77 toxicant flow was discontinued a f t e r 120 h. At the conclusion of the exposure i n Expt. 77 the water supply to the 0.63 mg/L tank was switched from FW to SW to determine whether. The surviving DHA-exposed f i s h would t o l e r a t e the a d d i t i o n a l osmotic s t r e s s . This r e s u l t would have to be taken i n t o consideration when choosing a "sublethal" exposure regimen f o r subsequent e l e c t r o l y t e studies. For the hypoxia experiment, sockeye salmon smolts were obtained from the Great Central Lake (Vancouver Island) run at the beginning of March 1978. Af t e r 2-1/2 months i n the outdoor holding tanks at PEI, 21 f i s h were transferred to each of 2 laboratory tanks by standardized methods pr e v i o u s l y described where they were kept f or 1 week p r i o r to the experiment. Well water temperature was maintained at 11.4 ±0.1 (X ±SE) during the 5-day exposure period to 0.65 mg/L DHA. A f t e r a 24 h st a r v a t i o n period, one tank was switched to hypoxic water (^  75% saturation) and the f i s h were given an a d d i t i o n a l 24 h acclimation to these conditions before the toxicant exposure was started. A f t e r 120 h, the toxicant exposure was discontinued, and the normoxic tank (90-95% saturation) was switched to hypoxic water to investigate the p o s s i b i l i t y of any l a t e n t s y n e r g i s t i c t o x i c i t y . F i s h s i z e during t h i s experiment was 13.7 ±0.17 cm and 26.3 ±1.09 g (X ±SE). 37 RESULTS AND DISCUSSION Behavioral Symptoms Frequent observations of f i s h during the course of the acute bioassays made i t possible to detect c e r t a i n behavioral changes induced by the toxicant exposure. The rate of progression of these behavioral a l t e r a t i o n s appeared to be dose dependent. Control f i s h maintained p o s i t i o n against the current i n a school which was centered under the shaded area of the tank, covering at most about 1/6th of the circumference of the donut. If disturbed, the school would immediately tighten up so that a l l the f i s h were under cover. At night, under the continuous dim i l l u m i n a t i o n provided, the s i z e of the school expanded to cover about 1/2 of the circumference with i n d i v i d u a l f i s h o ccassionally turning to swim with the current but r a r e l y for more than one c i r c u i t . In f i s h exposed to DHA, the f i r s t behavioral symptom to be observed was a reduction i n the compactness of the school r e s u l t i n g i n a gradual increase i n the sector occupied by the salmon. This schooling breakup occurred i n ^ 20 h i n f i s h exposed to 1 mg/L DHA. The normal cover response upon v i s u a l disturbance became progressively diminished u n t i l i t was t o t a l l y eliminated. At t h i s time a tap on the tank res u l t e d i n a somewhat confused e f f o r t to accelerate forward and school, but the f i s h appeared to be having problems with muscular coordination. At t h i s point, i n d i v i d u a l f i s h could no longer maintain p o s i t i o n against the current and began to d r i f t down-stream. At no point, however, d i d such f i s h abandon t h e i r r h e o t a c t i c response and appeared to be attempting to head upstream. A tap on the tank often r e s u l t e d i n a spasmotic, undirected movement. Eventually a f i s h manifesting t h i s behavior would lose equilibrium and be swept around the tank 38 with the current. Attempts at movement at t h i s stage res u l t e d i n muscular tremors and death followed approximately 3 h a f t e r equilibrium l o s s . I f the toxicant exposure was discontinued when the f i s h could no longer maintain s t a t i o n , they appeared to recover gradually and i n one case would accept food a f t e r ^ 24 h i n clean fresh water; however, no attempt was made to assess the recovery of f i s h which were at a more advanced stage of d e b i l i t y . The LC50 The r e s u l t s of the three acute bioassays are i l l u s t r a t e d i n F i g . 9 showing 96 h LC50's of 0.50, 0.79 and 0.88 mg/L DHA f o r Expts. 74, 76 and 77 r e s p e c t i v e l y . In Expt. 74, the shape of the t o x i c i t y curve suggests the presence of an acute t o x i c i t y threshold i n the 0.4 mg/L range; however the lowest concentration tested was 0.47 mg/L DHA. In Expt. 76 and 77 the t o x i c i t y curves generated by the l i m i t e d number of concentrations used remained i n the l i n e a r range. In Expt. 76 the toxicant exposure was continued u n t i l complete m o r t a l i t y occurred i n the lowest (0.65 mg/L) concentration; the l a s t f i s h died at 262 h. In Expt. 77, toxicant exposure was discontinued at 120 h and the water supply was switched from fresh to sea water (^  27 °/oo ) . In t h i s case the slope of the log p r o b i t l i n e used to determine the LT50^for the 0.79 mg/L group increased, suggesting a l a t e n t and enhanced t o x i c i t y brought on by the added s a l i n i t y s t ress. In the lowest concentration tested (0.63 mg/L), no m o r t a l i t y occurred during the 120 h exposure period; however, by the time the experiment was discontinued a f t e r 120 h i n sea water a further 6/14 f i s h had died. These r e s u l t s indicated that the p r i o r (sublethal) exposure to 0.63 mg/L. DHA reduced the subsequent s u r v i v a l of some sockeye salmon smolts i n clean sea water. There were no co n t r o l m o r t a l i t i e s i n any of the bioassays and i n Expt. 77 no behavioral changes could be detected i n co n t r o l f i s h which encountered the 3/ Time to 50% m o r t a l i t y (LT 50) 39 1 1 | I i ! ! J L 0.3 0.4 0.5 V 2 C o n c e n t r a t i o n of D H A m g / L Figure 9. T o x i c i t y curves i l l u s t r a t i n g 96 h LC50 values f or DHA to juvenile sockeye salmon_in f r e s h water. Confidence l i m i t s (95%) are given by O. rapid (.5.8 h f o r 95% replacement) FW->SW t r a n s i t i o n . The 96 h LC50 value of 0.50 mg/L DHA which was obtained i n Expt. 74 i s somewhat lower than that obtained i n Expt. 76 and 77. Measurements of dissolved oxygen during the course of Expt. 74 suggested an e l e v a t i o n i n oxygen consumption i n exposed f i s h . A f t e r 24 h of exposure, disso l v e d oxygen l e v e l s i n the tanks r e c e i v i n g the highest dose of DHA had dropped to 70% saturation while the controls remained above 90% saturation. Supple-mental aeration was added and d i s s o l v e d oxygen l e v e l s remained above 85% f o r the r e s t of the bioassay. Nevertheless, as the subsequent experiment showed that a reduction i n disso l v e d oxygen i n the water l e d to a marked increase i n DHA t o x i c i t y , the LC50 value of 0.50 mg/L DHA obtained during Expt. 74 was probably somewhat depressed by reduced 0 2. Expts. 76 and 77 were conducted with oxygen l e v e l s i n excess of 90% saturation and i n s p i t e of differences i n f i s h stock and s i z e , t e s t temperature and pH, the 96 h LC50's (0.79 and 0.88 mg/L) were remarkably close. E f f e c t s of Hypoxia The experiment confirmed the hypothesis of a j o i n t a c t i o n of low dissolved oxygen and DHA t o x i c i t y . F i s h exposed simultaneously to 0.65 mg/L DHA and reduced oxygen experienced 100% m o r t a l i t y while i n the group exposed at normal oxygen l e v e l s , only one f i s h died within the 120 h exposure period. These r e l a t i o n s h i p s are i l l u s t r a t e d i n F i g . 10 i n which the bottom heavy l i n e depicts measured D.O. l e v e l s , with the accompanying mortality curve (hypoxic) showing 50% m o r t a l i t y at 76 h. The r i s e i n D.O. l e v e l s from 78 to 120 h represents the reduction i n 0 2 consumption due to the reduction i n the number of f i s h and once the D.O. l e v e l reaches ^ 85% the m o r t a l i t y curve f l a t t e n s out somewhat due to a reduction i n the combined stress of hypoxia/toxicity. In the case of the group exposed under normal . 41 Figure 10. The e f f e c t of reduced dissolved oxygen i n the water on the t o x i c i t y of DHA (0.65 mg/L) to sockeye salmon smolts. D.O. conditions, a gradual increase i n oxygen consumption by these f i s h can be seen by the slope of the upper D.O. curve. The f i r s t m o r t a l i t y occurred when the D.O. l e v e l reached ^ 85% saturation. The sharp drop a f t e r 120 h represents the measurement 6 h a f t e r the toxicant flow had been discontinued and the water supply switched to hypoxic water. A second m o r t a l i t y occurred 3 h a f t e r the switch but no f u r t h e r m o r t a l i t i e s followed. This i n d i c a t e s that i t was the i n t e r a c t i o n of DHA with hypoxia which l e d to l e t h a l i t y . Once the toxicant was discontinued, the remaining f i s h appeared to recover well (behaviorally normal) even at 70% O2 saturation and began feeding again within 24 h. These r e s u l t s i n d i c a t e that hypoxia acted as a loading s t r e s s since a concentration of DHA shown previously to be sublethal under 0 2-saturated conditions proved l e t h a l when combined with a 30% hypoxia. This hypoxia i n i t s e l f was probably not deleterious to the salmon (Davis, 1976). In addition i t should be noted that sublethal DHA exposure i n a normoxic environment caused an increase i n the rate of removal of 0 2 from the water (dashed l i n e , Fig.10) i n d i c a t i n g an increase i n the oxygen demand of the f i s h . These observations compare favorably with the findings by Hicks and DeWitt(1971) of a marked increase i n the acute t o x i c i t y of whole KME to j u v e n i l e coho salmon at reduced l e v e l s of d i s s o l v e d oxygen. On the b a s i s of these experiments, 0.65 mg/L was chosen to represent a concentration of DHA which would be sublethal to j u v e n i l e sockeye salmon during an exposure period of 120 h i n well-oxygenated f r e s h water. Sub-sequent experiments were conducted to investigate the observed i n t e r a c t i o n between previous DHA exposure and s a l i n i t y s t r e s s . 43 B. EFFECTS OF ACUTE DHA EXPOSURE ON OSMOTIC BALANCE INTRODUCTION During the course of acute t o x i c i t y bioassays, some sockeye salmon appeared normal i n s i z e and shape while others developed a swollen or bloated appearance (Fig.11a). D i s s e c t i o n s of f i s h which were v i s i b l y swollen revealed an accumulation of f l u i d i n the stomach, i n some cases to such a degree that the organ was q u i t e t u r g i d ( F i g . l i b ) . As these symptoms were suggestive of a water balance problem, experiments were conducted to quantify t h i s response to DHA poisoning. Condition f a c t o r s weight (K = r"~-3 x 100) were c a l c u l a t e d as a measure of body "fatness" length- 3 • (Lagler, 1969) and measurements of t o t a l body water were made to determine whether a general hydration was occurring. Following these preliminary observations, experiments were conducted to e s t a b l i s h whether t h i s apparent osmotic imbalance extended to the muscle t i s s u e . This being the case, the muscle of f i s h exposed to DHA i n f r e s h water should gain weight (hydrate), whereas f i s h exposed i n sea water should lose water (dehydrate). MATERIALS AND METHODS Bloa t i n g During a preliminary bioassay (Expt. A) i n which a wide range of f i s h s i z e was used to i n v e s t i g a t e the r e l a t i o n s h i p between f i s h s i z e and acute DHA t o x i c i t y (1.1 mg/L),the bloated stomachs of f i v e v i s i b l y swollen sock-eye salmon ( f i s h s i z e range, 11.2 to 369 g) were excised by a cut d i s t a l to both sphincters. Each f l u i d - f i l l e d sac was then d r i e d i n a f o r c e d - a i r oven at 105°C and the % water was compared to that of stomachs taken from f i s h which had died during the bioassay but which had maintained an apparently normal body form. Figure 11. I l l u s t r a t i o n of the swelling of sockeye salmon caused by DHA exposure. Condition factors were calculated f o r f i s h which had died i n three preliminary bioassays CExpts. B, C and D): In addition, t o t a l body water was measured i n Expt. D. Experimental d e t a i l s are outlined i n Tables III and IV i n the Results section. Muscle Water Following these preliminary observations, the percentage muscle water was determined i n f i s h exposed to DHA i n fresh water (Expt. E) and i n sea water (Expt. F ) . In Expt. E, underyearling sockeye salmon were exposed to DHA i n fresh water and each f i s h was c o l l e c t e d at the stage when i t could no longer maintain rheotaxis i n the annular tanks. In the second experiment, underyearling chum salmon were exposed to DHA i n sea water. Half of the f i s h were c o l l e c t e d at death, while the other h a l f were s t i l l a l i v e when the bioassay was discontinued. The sockeye salmon were of the Great Central Lake (Vancouver Island) stock and were r a i s e d from f e r t i l i z e d eggs at PEI. At the time of the experiment, these f i s h were 8 months o l d . As no sea-water adapted sockeye salmon were a v a i l a b l e , chum salmon from the Inches Creek stock (Dewdney, B.C.) were used and had been hatched at PEI where they were maintained i n sea water (27-29 °/oo) . In the case of survivors or d r i f t i n g f i s h , sampling was done by gently dipnetting i n d i v i d u a l s as they d r i f t e d around the tank. F i s h were k i l l e d by a sharp f l i c k of the fi n g e r , b l o t t e d and a t i s s u e sample was taken f o r determination of % water. A transverse cut was made through the body immediately p o s t e r i o r to the vent, with a second cut about 1 cm further p o s t e r i o r l y . The bulk of such a trunk section consisted of muscle but included several vertebrae and skin. The sample was weighed i n an aluminum dish, d r i e d as described previously, and the moisture content was expressed as % muscle water. RESULTS AND DISCUSSION Not a l l f i s h exposed to DHA developed a bloated appearance nor was the swelling a post-mortem development as i t was frequently observed many hours before equilibrium l o s s i n acute bioassays. In Expts. B, C, and D, condition factors of sockeye salmon which died during DHA exposure are s i g n i f i c a n t l y higher than the controls (Table III and IV). The progressive nature of t h i s weight gain i s r e f l e c t e d by a gradual increase i n K factor before and a f t e r equilibrium l o s s leading to death (Table I I I , footnote 2). Of the 24 f i s h exposed to DHA during Expt. A, 5 had v i s i b l y swollen abdomens. The accumulation of f l u i d i n the stomachs of these f i s h i s shown i n the r e s u l t s i n Table V. The stomach of one f i s h which appeared p a r t i a l l y swollen contained an amount of water mid-way between that present i n normal and swollen f i s h . In Expt. D, t o t a l body water measurements confirmed that DHA exposed f i s h contained s i g n i f i c a n t l y more water than the controls (Table IV). These experiments showed that i n some salmon DHA exposure i n fresh water r e s u l t s i n a dramatic increase i n water ingestion, leading to a v i s i b l e accumulation of water and that i n others, although the swelling i s not apparent, there occurs a general hydration which i s r e f l e c t e d by elevat-ed K-factors and increased t o t a l body water content. The muscle water experiments described below were conducted to determine whether t h i s edematous condition extended to the ti s s u e s of f i s h exposed to DHA. Muscle Water The r e s u l t s of the sockeye salmon experiment are presented i n Table VI and show that exposure to DHA r e s u l t e d i n a hydration of muscle t i s s u e i n f resh water. As these changes were observed at a time when the f i s h could no longer maintain p o s i t i o n against the current perhaps t h i s muscle 47 Table I I I . Condition f a c t o r s of underyearling sockeye salmon exposed to acutely l e t h a l and su b l e t h a l concentrations of DHA i n f r e s h water (Expts. B and C). Mean ±SE (DHA) length weight c o n d i t i o n ^ f a c t o r Bioassay mg/L N cm g K 1.81 .8 10.8 ±0.26 17.4 ±1.11 1.37*±0.03 0.83 7 10.3 ±0.23 15.2 ±1.14 1.36*±0.02 C o n t r o l 9 10.9 ±0.16 15.6 ±1.09 . 1.07 ±0.02 C 2 ' 1.32 9 11.9 ±0.65 22.7 ±1.13 1.35*±0.02 0.573 ' 6 11.2 ±0.18 19.0 ±0.91 1.37*±0.02 C o n t r o l 10 11.8 ±0.33 17.9 ±1.43 1.08 ±0.03 1 weight , X K = - ^-7,3 x 100 length 2 cm g one f i s h sampled before l o s s of e q u i l i b r i u m 12.0/19.6 K=1.13 one f i s h sampled a f t e r l o s s of e q u i l i b r i u m 11.8/20.5 K=1.25 * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p<0.05 Student's t - t e s t 3 Time to 50% mortality (LT 50)=15.3 days Table IV. Condition f a c t o r and t o t a l body water i n underyearling sockeye salmon which died during exposure to 0.95 mg/L DHA i n f r e s h water (Expt. D). length weight c o n d i t i o n body water N cm g f a c t o r K % Exposed 19 9.6 ±0.24 9.2 ±0.60 1.04* ±0.02 78.55*10.35 Control 10 10.2 ±0.20 9.0 ±0.55 0.85 ±0.01 75.16 ±0.88 1 LT 50 = 62.5 h * s i g n i f i c a n t l y d i f f e r e n t from controls p<0.05. Student's t - t e s t Table V. Percentage water of stomachs diss e c t e d from "swollen" and "normal" salmon exposed to 1.11 mg/L DHA (Expt. A). N % Water (Mean ±SE) Swollen ( 5) 91.7*±1.2 Normal (10) 79.0 ±1.7 P a r t l y Swollen ( 1 ) 84.8 * s i g n i f i c a n t l y d i f f e r e n t from controls p<0.05. Student's t - t e s t Table VI. Muscle water i n underyearling sockeye salmon exposed to DHA1 i n fresh water and sampled at the d r i f t i n g stage. Mean ±SE Length Weight Muscle Water N cm g % Exposed 20 11.2 ±0.27 13.96 ±0.94 77.05*±0.31 Control 20 11.3 ±0.24 12.89 ±0.85 75.78 ±0.14 ^1.4 mg/L * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p<0.05. Student's t - t e s t 51 hydration i n t e r f e r e d with normal c o n t r a c t i l e processes and thus contributed to a reduction i n swimming performance. The r e s u l t s of the exposure of chum salmon to DHA i n sea water are shown i n Table VII and i l l u s t r a t e a s i g n i f i c a n t dehydration of muscle tis s u e when compared to the controls. The gradual development of dehydration i s i l l u s t r a t e d by the reduction i n water content of salmon which had survived the 96 h exposure to DHA. This value suggests that the amount of dehydration increases with exposure time. These preliminary experiments indicated that exposure to DHA l e d to an osmotic imbalance i n a d i r e c t i o n dictated by the osmotic gradient between body f l u i d s and the surrounding water. As such an osmotic imbalance could be accompanied by an i o n i c disturbance, experiments were conducted to determine plasma i o n i c composition i n sockeye salmon exposed to DHA i n fresh water. Subsequently, experiments were performed to measure the hydromineral regulatory a b i l i t y of salmon exposed s u b - l e t h a l l y to DHA and then trans-f e r r e d to sea water. Table VII. Muscle water i n underyearling chum salmon exposed to DHA-*- i n sea water. Mean ±SE Length Weight Muscle Water N cm g % At death 10 10.0 ±0.23 7.70 ±0.53 77.09* ±0.36 Exposed Survivors 10 10.0 ±0.16 7.62 ±0.43 79.53*±0.38 Controls 20 10.1 ±0.13 7.92. ±0.33 80.72 ±0.14 1 1.4 mg/L * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p<0.05. Student's t - t e s t PART I I . PRINCIPAL EXPERIMENTS A. EFFECTS OF SUBLETHAL DHA EXPOSURE ON HYDROMINERAL BALANCE IN SOCKEYE SALMON SMOLTS INTRODUCTION The suggestion made from the previous experiments that osmotic imbalance was also accompanied by an e l e c t r o l y t e disturbance was confirmed i n an exploratory experiment (unpublished observations) i n which blood e l e c t r o l y t e l e v e l s were measured i n sockeye salmon s u b l e t h a l l y exposed to DHA i n f r e s h water. Exposure to DHA res u l t e d i n a s i g n i f i c a n t reduction i n the concen-t r a t i o n s of the main plasma e l e c t r o l y t e s Na + and C l . However, no changes + ++ ++ were observed i n plasma K or Mg l e v e l s and plasma Ca • concentrations increased. This complex response cannot be explained by a simple DHA-induced hydration. To investigate these findings more f u l l y , t h i s experiment was repeated and the observations were also extended i n t o the seawater phase. As discussed i n the General Introduction, p r i o r sublethal DHA exposure followed by the movement of f i s h i n t o sea water was meant to simulate the exposure of sockeye salmon to pulp m i l l waste i n a r i v e r during the course of smolt migration. Under these conditions, the extent of toxicant exposure would be l i m i t e d to the migration time i n f r e s h water and subsequent entry i n t o the sea would involve a rapid t r a n s i t i o n from a hypotonic to a hypertonic medium. Although t h i s seawater acclimation period comprises a "recovery phase", as the toxicant exposure has been discontinued, the i n t e r a c t i o n of the e f f e c t s of p r i o r exposure with natural s a l i n i t y stress could have an e f f e c t on hydro-mineral homeostasis. Three experiments were conducted (Expts. 1,2 and 3) i n which sockeye salmon smolts were exposed to a sublethal dose of DHA i n fresh water and subsequently "transferred" to sea water. Osmoregulatory performance was 54 gauged by measuring plasma e l e c t r o l y t e concentrations at the end of the toxicant exposure period and, subsequently, by following the time course of plasma e l e c t r o l y t e regulation during the t r a n s i t i o n to sea water. In view of the r e s u l t s obtained i n fresh water, i t was hypothesized that DHA exposure should lead to elevated plasma i o n i c l e v e l s i n f i s h i n sea water i f the toxicant acted to cause a l o s s i n ionoregulatory p r e c i s i o n . During Expt. 1 however, some of the f i s h were suspected of s u f f e r i n g from a chronic i n f e c t i o n of b a c t e r i a l kidney disease (BKD) and the presence of the i n f e c t i o n was confirmed during Expt. 2 which followed immediately. B a c t e r i a l kidney disease involves a gradual destruction of the kidney which i s intimately involved with hydromineral balance i n f i s h . Thus, to reduce the p o s s i b i l i t y of the disease confounding the e f f e c t s caused by the toxicant, i t seemed imperative to devise a procedure to eliminate from the data f i s h i n which the i n f e c t i o n had reached an advanced stage; t h i s was done using hematocrit values. Blood hematocrit i s known to drop gradually as the disease progresses, therefore data c o l l e c t e d from salmon with abnormally low hemato-c r i t s were deleted. A thorough discussion of b a c t e r i a l kidney disease and i t s i n t e r a c t i o n with DHA t o x i c i t y , as w e l l as the d e t a i l s of the screening method and tables containing the deleted data are presented i n Appendix I I . At a l a t e r date, when a new stock of disease-free salmon became a v a i l a b l e , the e l e c t r o l y t e balance experiment was repeated a t h i r d time (Expt. 3) and confirmed the r e s u l t s which had been obtained i n Expts. 1 and 2. MATERIALS AND METHODS Three s i m i l a r but separate experiments were conducted i n which sockeye smolts were exposed to 0.65 mg/L DHA f o r 5 days (120 h) i n f r e s h water under continuous flow conditions. At the end of t h i s sublethal exposure period, toxicant administration was discontinued, and the f r e s h water supply was 55 switched to sea water. A 95% replacement i n 5.8 h yie l d e d a s a l i n i t y of ^ 26 °/oo at the end of t h i s time. The f i r s t blood sampling was conducted at the end of the freshwater DHA exposure peri o d (G h) and subsequently f o r 120 h at i n t e r v a l s of 24 h to follow the time course of adaptation to sea water. F i s h handling, blood sampling and an a l y s i s p r o t o c o l was performed as described i n the General Methods section. The s i z e s of the f i s h used i n the three experiments are given i n Table VIII. The three experiments were conducted using two separate stocks of sockeye salmon. Expts. 1 and 2 were performed i n May 1977 u t i l i z i n g f i s h which were obtained as yearlings from the Cultus Lake stock and had been kept at PEI f o r 6 months p r i o r to use. This was the stock o f f i s h which developed b a c t e r i a l kidney disease. In June 1977, sockeye smolts from the Great C e n t r a l Lake (Vancouver Island) run were brought to PEI but proved too small to conduct the corroborative experiment (Expt. 3). As a r e s u l t , Expt. 3 was conducted i n the f i r s t week of December 1977 at which time these f i s h were 21 months o l d . Thus while the s i z e of f i s h used i n the three experiments was s i m i l a r , the sockeye i n Expt. 3 were of a d i f f e r e n t stock and were three months older. RESULTS Latent Acute T o x i c i t y For the purposes of these experiments, "sublethal" was defined as a dose (the product of concentration x time) causing n e g l i g i b l e m o r t a l i t y i n 120 h. According to t h i s d e f i n i t i o n 0.65 mg/L DHA was sublethal i n Expts. 1 and 3. In Expt. 2 however, mo r t a l i t y reached % 7% during the exposure period. This increase i n t o x i c i t y was caused by the BKD i n f e c t i o n and i s discussed i n Appendix I I . •J Table VIII. Size of the sockeye salmon used i n the e l e c t r o l y t e balance experiments: (Expts. 1,2 and 3) . Mean* SE(n) Time Control E * P ° s e d in Fork length Wet weight Fork length Wet weight hours cm g cm Experiment 1 0 14 9 0 38(10) 33 97 2 66 14 9 0. 27(10) 34 42 1 83 24 15 0 0 47( 9) 33 93 3 24 14 3 0 21(10) 30 71 2 06 48 14 8 0 30(10) 32 16 1 96 14 2 0 50( 7) 29 44 2 75 72 14 6 0 27 ( 9) 31 21 1 98 15 0 0 26( 8) 33 43 1 69 96 15 2 0 48{ 9) 34 61 3 54 15 1 0 28( 9)- 34 02 1 92 120 14 9 0 22(10) 32 48 1 53 15 1 0 40( 7) 35 53 '3 23 Experiment 2 0 15 7 0 38(10) 40 93 2 33 15 9 0 30(11) 40 65 2 27 24 16 4 0 32( 9) 44 09 2 43 15 4 0 33( 9) 37 06 2 66 48 15 8 0 60 ( 6) 40 82 5 25 15 3 0 32(10) 36 75 2 10 72 15 .5 0 30 ( 8) 37 06 2 43 15 6 0 24 ( 7) 37 36 1 78 96 15 8 0 33(10) 39 85 2 19 16 0 0 39( 8) 39 69 2 97 120 15 6 0 40 ( 8) 38 15 2 46 15 3 0 42 ( 8) 35 90 3 18 Experiment 3 0 15 9 0. 35(11) 36. 68 2 69 15 3 0. 33(12) 31 74 2 18 24 15 0 0. 37(11) 29. 07 2 25 15 3 0 41(12) 32 09 2 58 48 15 8 0 35(12) 33. 71 2 10 15 6 0 47(11) 32 56 2 87 72 16 0 0 24 ("ll) 35 44 1 75 15 5 0 52(10) 32 48 3 79 96 15 2 0 45(12) 32 14 2 70 16 0 0 42(10) 36 15 2 56 120 16 0 0 32(12) 36 04 2 58 15 6 0 43(12) 34 28 2 30 57 The presence of a l a t e n t acute t o x i c i t y of DHA became apparent when the previously exposed f i s h were faced with a s a l i n i t y challenge. As i l l u s t r a t e d i n Fig.25 (p.155) t h i s combination of stressors led to m o r t a l i t i e s i n Ex p t . l only a f t e r the f i s h had been i n sea water f o r 24 h. In Expt. 2, m o r t a l i t i e s which s t a r t e d during the freshwater exposure period appeared to continue at the same r a t e f o r the f i r s t 24 h i n sea water, with a subsequent break i n the slope of the t o x i c i t y curve i n d i c a t i n g an attenuation of l a t e n t acute t o x i c i t y a f t e r 48 h i n clean sea water. Actual m o r t a l i t i e s f o r Expts. 1, 2 and 3 were 7/60, 15/75 and 2/72. These f i g u r e s include "moribund" f i s h f o r Expt. 2 but do not include the 3 " d r i f t e r s " i n Expt. 3-which were sampled i n the e a r l y stages of i n t o x i c a t i o n . "Moribund" and " d r i f t e r " f i s h are discussed i n the l a s t p a r t of the Results section. Control m o r t a l i t i e s were n e g l i g i b l e , with only 1 of the 204 t o t a l dying of what appeared to be a fungus i n f e c t i o n of the g i l l s . Sublethal E f f e c t s Behavioral Observations Observations made during the course of the toxicant exposure i n d i c a t e d a s l i g h t reduction i n the compactness of the school, a behavior which corresponds to the f i r s t stage of the sequence described i n the Behavioral Symptoms po r t i o n of the Acute T o x i c i t y section (p. 37 ) . The normal response to v i s u a l disturbance (cover response) was only s l i g h t l y reduced i n Expt. 1 and 3 whereas i n Expt. 2 the f i s h generally reacted more slowly. In Expt. 2, 15 f i s h were more severely a f f e c t e d a f t e r 103 h exposure; these were sampled p r i o r t o the end of the exposure peri o d and are discussed i n a l a t e r s e c t i o n as "moribund" f i s h . 58 This slowness i n response of DHA-exposed f i s h generally became more pronounced during the f i r s t 24 h of seawater adaptation, and many f i s h s t i l l d i d not display a normal cover response a f t e r 72 h i n sea water. In contrast the behavior of co n t r o l f i s h remained unchanged throughout the en t i r e exper-iment . Hydromineral Balance i n Fresh Water The plasma e l e c t r o l y t e l e v e l s i n sockeye salmon smolts exposed f o r 120 h to 0.65 mg/L DHA.in fresh water are presented i n Table IX and i l l u s t r a t e d i n Fig.12. In addition to plasma ions, blood hematocrit was determined i n a l l three experiments whereas % muscle water was measured only i n Expts. 2 and 3. Table IX gives the means of the actual values measured i n each of the three experiments, whereas Fig.12 combines these means to i l l u s t r a t e an o v e r a l l "average response". S t a t i s t i c a l s i g n i f i c a n c e shown i n Fig.12 was determined by combining the p r o b a b i l i t i e s obtained from separate te s t s of s i g n i f i c a n c e between "control" and "exposed" means i n each experiment. The Student's " t " t e s t was used and was corrected when variances were unequal. Exact p r o b a b i l i t i e s were determined f o r each c a l c u l a t e d " t " and then pooled according to the method given i n Sokal and Rohlf (1969). This treatment provides a sin g l e t e s t of s i g n i f i c a n c e of the aggregate based on the product of the prob-a b i l i t i e s observed i n d i v i d u a l l y (Fisher, 1958). This approach was used because, although the three experiments were s i m i l a r i n design, they were not i d e n t i c a l , so that pooling of a l l the data f o r j o i n t s t a t i s t i c a l treatment was not warranted. Fig.12 shows that a 120 h exposure to a sublethal dose of DHA caused a s i g n i f i c a n t disturbance i n the l e v e l s of a l l plasma e l e c t r o l y t e s investigated, except f o r sodium. The d i r e c t i o n of the change however, was not uniform. 59 27X3 275 2 6 0 2 8 5 ~l 1 — I ' — | — I — 1 — I — I — I — I — I — I — I — | — I — r C O N T R O L E X P O S E D 2 9 0 m O s m / k g -r~l 11TJ 115 120 125 C O N T R O L E X P O S E D Osmolality ! 3 0 m E Q / L " T — I — | — I — I — I — 1 — | I I — I — I — | 1—I—I—I—| 1 ) •• Chloride 1 4 0 1 4 5 1 5 0 1 5 5 1 6 0 C O N T R O L E X P O S E D I I I I 1 1 1 1 1~\ 1 1 1 1 1 1 1 I 1 1 mEq/L J Sodium 2 . 5 3 . 0 3 . 5 C O N T R O L E X P O S E D 1 1 1 • i « 1 mEq/L Potassium 5 0 5 . 5 6 . 0 C O N T R O L E X P O S E D mEq/L I 1 1 1 | 1 1 1 1 1 C a l c i u m 1 .0 1 . 5 C O N T R O L ] E X P O S E D ~~i r T 1 1 r 2 0 mEq/L Magnesium 3 0 3 5 4 0 4 5 5 0 % 1 — I — I — I — | — I — I — < — I — p ~ 1 — l — I — I — | — I — I — I — I — | C O N T R O L E X P O S E D Hematocrit C O N T R O L E X P O S E D 7 5 7 6 7 7 7 8 7 9 8 0 T | I | I | I | ' | 1 Muscle Water Figure 12. Plasma e l e c t r o l y t e l e v e l s , hematocrit and muscle water content i n sockeye salmon exposed to 0.65 mg/L DHA for 120 h i n fresh water. Values are means f o r each parameter based on Expts. 1, 2 and 3 except f o r muscle water which was measured i n Expt. 2 and 3 only. S i g n i f i c a n c e l e v e l p<0.05^; p<0.'01#4 60 Table IX. Plasma i o n i c composition, hematocrit and muscle water content of sockeye salmon exposed to 0.65 mg/L DHA for 120 h i n fresh water i n Expts. 1, 2 and 3. Osmolality mOsm/kg Experiment 1 Control 286.6 1.77(10) Exposed 276.4* i.63( 9) Experiment 2 287.6 3.11(10) 286.0 3.07(10) Experiment 3 285.1 3.23(11) 273.8 2.13(12) Chloride mEq/L Control 124.8 0.39(10) Exposed 114.4* 1.35(10) 123.9 1.51(10) 114.5 a 1.00(11) 124.3 1.15(11) 110.6* 1.40(12) Sodium mEq/L Control 153.83 1.31(10) Exposed 152.01 1.71(10) 147.29 1.13(10) 146.08 2.03(11) 151.94 2.37(11) 154.14 1.61(12) Potassium mEq/L Control Exposed 2.81 0.21(10) 1.93° 0.24(10) 3.63 0.16(10) 3.56 0.16(11) 3.20 0.14(11) 2.68 0.26(12) Calcium mEq/L Control Exposed 5.69 0.12(10) 6.18 0.15(10) 5.38 0.07(10) a 6.18 0.07(11) 4.29 0.08(11) 4.78 0.21(12) Magnesium mEq/L Control Exposed 1.50 0.03(10) 1.52 0.04(10) 1.55 0.03(10) b 1.74 0.04(11) 1.37 0.05(11) 1.60 a 0.03(12) Hematocrit Muscle Water % Control 33.97 1.63(10) 36.30 1.12(10) 40.56 0.73(11) Exposed 45.00 2.00(10) Control Exposed 44.76 1.19(11) 74.79 0.42(10) 75.69 0.18(11) 47.83 1.04(12) 79.50 0.53(11) 81.17 0.59(12) Total sum of ions a p<0.001, b p<0.01, ° p<0.02, d p<0.05 d i f f e r s s i g n i f i c a n t l y from co n t r o l t - t e s t 61 O v e r a l l , DHA exposure i n f r e s h water l e d to most pronounced and consistent changes i n plasma l e v e l s of C l and C a + + and i n blood hematocrit. In a l l ++ ++ + three experiments, plasma Ca and Mg increased whereas plasma Na l e v e l s remained remarkably stable. The decrease i n plasma osmolality was accompanied by an increase i n muscle water l e v e l s . Hydromineral Balance i n Sea Water (Plasma E l e c t r o l y t e s ) The e f f e c t s of the combined stress of p r i o r toxicant exposure and s a l i n i t y on plasma e l e c t r o l y t e s of sockeye salmon are presented i n Table X and i l l u s t r a t e d i n Fig.13.Table x gives the means of the various plasma e l e c t r o l y t e determinations f o r Expts. 1, 2 and 3, while Fig.13 provides a comparison of "exposed" r e l a t i v e to "c o n t r o l " values; controls are shown as 100. Each point represents an average of the combined responses obtained i n Expts. 1, 2 and 3, while the l e v e l of s t a t i s t i c a l s i g n i f i c a n c e of the departure of each point from the c o n t r o l (dotted l i n e ) was determined from pooled p r o b a b i l i t i e s as described previously. S i g n i f i c a n c e l e v e l s are shown i n the box below each graph. Note that the points f o r 0 hours SW represent the various e l e c t r o l y t e values a f t e r 120 h DHA exposure i n fresh water. Since they are not part of the seawater phase, for reasons of c l a r i t y they are not connected to the curves but have been shown to provide a r e l a t i v e i n d i c a t i o n of the magnitude of the d i r e c t e f f e c t of DHA on e l e c t r o l y t e balance i n fresh water. The r e s u l t s summarized i n Fig.13 show that the balance of a l l the plasma e l e c t r o l y t e s except f o r K + was a l t e r e d i n f i s h which had previously been exposed to DHA. The departures from the co n t r o l values were a l l i n the p o s i t i v e d i r e c t i o n and subsequently returned to normal l e v e l s a f t e r varying i n t e r v a l s . Plasma C l concentrations remained elevated (p<0.001) f o r 96 h, 62 Figvre 13. The change in plasma electrolyte levels in sockeye salmon during adaptation to sea water following a 120 h exposure to 0.65 mg/L DHA in fresh water. Points for esch parameter represent the overall mean % change (relative to a control value of ICQ) derived from Experiments 1, 2 and 3. The level of statistical significance of the change is given at the base of each graph. Table X. Plasma e l e c t r o l y t e l e v e l s [Mean -SE(N)] i n sockeye salmon during sea-water adaptation following a 120 h exposure to 0.65 mg/L DHA i n fresh water i n Expts. 1, 2 and 3. EXPT 1 T i n e i n sea water hours Osmolality mOsjn/kij Exposed Chloride mEq/l. Exposed Sodium roEq/L Exposed POt&SSluM nEq/L Control Expoaod calcium nEq/L Bf-q/L Exposed 0 266 6 1 77(10) 276 4 " l 6J( 9) 124 U 0.39(10) 114 A 4 1 35(10) 153.83 1 11(10) 152 01 1. 71(10) 2. 81 0 21(10) 1 93 C 0 24(10) 5 69 0 12110) 6 18" 0 15(10) 1 50 0.03110) 1 52 0.04(10) 24 299 5 1 641 8) 141 2 2 061 5) 128 3 0.041 9) 146 7° 2 86(10) 162.36 2 49 ( 9) 179 75* 3. 33(10) 1. 60 0 30 ( 9) 1 84 0 17(10) 6 22 0 151 9) 7 15' 0 22(10) 2 19 0.081 9) 1 62* 0.26(10) 48 291 1 3 40 ( 9) 316 7 10 69 ( 6) 127 2 1.18(10) 137 1* 3 06( 7) 154.26 0 92(10) 165 d 16 4. 211 7) 2. 42 0 35(10) 2 83 0 241 7) 5 94 0 06110) 6 26 0 19( 7) 1 89 0.04(10) 3 53d 0.491 7) 72 299 1 3 59 ( 7) JOB 3 4 38 ( 8) 133 2 1.48( 9) 130 8 1 131 8) 159.44 2 23( 9) 164 72 2. 89 ( 8) 3 95 0 141 9) 3 41 0 201 8) 5 88 0 07( 9) 5 91 0 141 8) 1 99 0.081 9) 2 30 0.191 8) 96 295 1 2 06( 9) 293 1 2 091 9) 131 3 1.641 9) 130 9 1 5BI 9) 160.65 2 21 (9) 155 30 1. 51 (9) 3. 44 0 09( 9) 3 62 0 28( 9) 5 86 0 07( 91 5 83 0 07( 9) 1 47 0.091 9) 1 92 0.301 9) 120 29 5 4 2 48(10) 299 9 1 85( 7) 129 7 0.34(10) 111 4 0 57( 7) 160.82 1 09(10) 161 56 2. 10( 7) 3. 67 0 11(10) 3 75 0 111 7) 5 89 0 08(10) 5 99 0 091 7) 1 66 0.04(10) 1 72 0.041 7) EXPT 2 0 287 6 3 11(10) 286 0 3 07(10) 123 9 1.51(10) 114 a 5 1 00111) 147.29 1 13(10) 146 08 2.03111) 3.63 0 16(10) 3 56 0 16(11) 5 38 0 07(10) 6 a 18 0 07(11) 1 55 0 03110) 1 7 ? 0.04(11) 24 296 2 2 301 9) 325 5 b 6 92 ( 8) 130 3 1.781 9) 142 b 1 2 78( 8) 155.90 1 55( 9) 170 21* 2.891 8) 3.39 0 201 9) 3 85 0 141 8) 5 52 0 071 9) 5 d 94 0 151 8) 1 94 0 091 9) 3 i e " 0.37( 8) 48 306 0 3 72( 6) 305 6 2 64 ( 9) 132 4 0.911 6) 135 0 0 95(10) 160.13 2 84 ( 6) 165 09 1.67(10) 3.31 0 181 6) 3 80 0 20(10) 5 45 0 26( 61 5 52 0 04(10) 1 83 0 04 ( 6) 2 22 0.17(10) 72 289 4 2 68 ( 7) 314 o* 3 27( 6) 128 6 1.001 8) 140 3° 1 86( 6) 154.46 1 831 8) 165 b 65 3.111 6) 4.01 0 21( 8) 4 17 0 25 ( 6) 5 73 0 331 7) 5 56 0 OBI 6) 1 84 0 061 8) 2 92 0.461 6) 96 293 8 3 55(10) 301 1 2 09 ( 7) 129 0 1.11(10) 135 l " 0 84 ( 8) 155.04 1 74(10) 157 51 4.2K 8) 3.61 0 18(10) 3 60 0 151 8) 5 69 0 10(10) 5 48 0 161 8) 1 65 0 05(10) 1 63 0.071 8) 120 295 0 2 24 ( 8) 294 0 1 91 ( 8) 129 8 0.861 8) 129 3 1 30 ( 0) 152.03 1 921 8) 152 49 1.611 8) 4.02 0 19( 8) 3 94 0 181 8) 5 68 0 141 8) 5 54 0 091 8) 1 64 0 051 B ) 1 62 0.051 6) EXPT 3 0 285 1 3 23(11) 273 b 8 2 13(12) 124 3 1 15(11) 110 6* 1 40(12) 151.94 2 37111) 154 14 1.61(12) 3.20 0 14(11) 2 68 0 26(12) 4 29 0 08111) d 4.78 0 21112) 1 37 0 05(11) 1 a 60 0.03(12) 24 290 2 2 07(11) 325 9* 7 00(12) 128 6 1 20(11) 146 3* 3 29(12) 151.76 1 21(11) 168 71* 3.58(12) 3.15 0 22(11) 3 19 0 16(12) 4 70 0 12(11) c 5.18 0 13(121 2 00 0 14(11) 2 52C 0.14112) 48 291 7 1 57(12) 305 d 6 5 57(11) 129 3 1 13(12) 137 5° 2 56(11) 153.15 0 95(12) 157 54 3.11(11) 3.28 0 18(12) 3 48 0 14(11) 4 SO 0 06(12) 4.57 0 09111) 1 72 0 09(12) 2 53b 0.18111) 72 299 0 3 17(11) 312 6 8 30(10) 130 5 2 22(11) 139 d 6 3 70(10) 158.44 1 86(11) 161 65 4.19(10) 3.40 0 14(11) 4 14 C 0 23(10) 4 99 0 14(11) 4.406 0 12(101 1 71 0 04111) 2 83 d 0.47(10) 96 288 6 2 37(12) 300 3 3 00(10) 129 2 1 49(12) 136 b 3 1 47(10) 149.33 1 19(12) 153 51 2.14(10) 3.69 0 14(12) 3 88 0 23(10) 4 61 0 08(12) 4.27° 0 11(10) 1 78 0 08(12) 2 43 0.39110) 120 292 3 2 09(12) 291 7 1 67(12) 130 7 1 55(12) 131 5 0 78(12) 151.87 0 87(12) 149 22 1.33(12) 3.48 0 15(12) 3 61 0 30(12) 4 38 0 07(12) 4.08* 0 07(12) 1 73 0 06(11) 1.76 0.11(12) S i g n i f i c a n c e L e v e l P < 0.001 0.0I 0.02 0.05 64 Na and Mg f o r 72 h (p<0.05) while plasma Ca l e v e l s had returned to the con t r o l range within 48 h. Of a l l the e l e c t r o l y t e s investigated plasma Mg + + increased by f a r the most, reaching a maximum average of 3.10 mEq/L within 24 h as compared to 2.04 mEq/L f o r controls within 24 h (Table X). Plasma Mg + + l e v e l s s t i l l remained elevated at the 96 h sampling period however, the gradual return to cont r o l l e v e l s coupled with a high v a r i a t i o n i n the exposed groups rendered t h i s d i f f e r e n c e s t a t i s t i c a l l y n o n - s i g n i f i c a n t . In general, DHA exposure reduced the p r e c i s i o n of regulation as•indicated by an increase i n the standard errors i n Table X and i l l u s t r a t e d i n F i g . 16 fo r Expt. 3. O v e r a l l , these changes l e d to a general increase i n the t o t a l plasma osmolality which p e r s i s t e d f o r 72 h a f t e r toxicant exposure. Hydromineral Balance i n Sea Water (Hematocrit, Muscle Water, Gut_Water_Content) In addition to the determination of plasma e l e c t r o l y t e concentrations, measurements were made of blood hematocrit, muscle water and gut water content. I f the fresh water DHA exposure led to a subsequent dehydration of salmon i n sea water, t h i s could be r e f l e c t e d i n a lowering of muscle water content coupled with an increase i n the rate of sea water ingestion as a counter-measure . Blood hematocrit l e v e l s at the various sampling periods are presented i n Table XI and a comparison between exposed arid c o n t r o l groups i s i l l u s t r a t e d i n F i g . 14 (top). Hematocrit was measured i n Expts. 1, 2 and 3, whereas muscle water was determined i n Expt. 2 and 3. The % water i n the gut was measured only i n Expt. 2; the r e s u l t s are presented i n Table XII and are i l l u s -t r a t e d i n F i g . 15 which also shows the r e l a t i o n of muscle and gut water determinations i n "moribund" salmon to those of co n t r o l and exposed groups. Table XI. Hematocrit of sockeye salmon a f t e r a 5-day exposure to 0.65 mg/L DHA i n fresh water followed by exposure to sea water i n Expts. 1, 2 and 3. Hematocrit % Time in sea water Control Exposed n Mean ± SE (n) Mean * SE (n) Experiment 1 Experiment 2 0 33. 97 1. 63(10) 45. 00 2. oo(io) a 24 45. 19 2. 19 ( 9) 49. 36 2. 67(10) 48 42. 12 1. 06(10) 41. 74 1. 24( 7) 72 38. 33 0. 89( 9) 36. 18 0. 63 ( 8) 96 35. 59 0. 96( 9) 35. 57 1. 22( 9) 120 36. .58 1. ,06(10) 34. .63 0. 85( 7) 0 36. • 3 0 1. .12(10) 44. .76 1. .19(11) 24 38, .99 0. .85( 9) 38. .34 2 • 01( 8) 48 37 .28 2 .26( 6) 36. .53 1 .47(10) 72 35 .05 1. .84( 8) 31 .93 1 .26( 7) 96 35 .35 1 .46(10) 37 .75 1 .38( 8) 120 36 .73 1 -76( 8) 31 .60 1 .44( 7) b Experiment 3 0 24 48 72 96 120 40.56 0.73(11) 44.80 1.08(11) 41.28 1.10(12) 41.86 0.90(11) 40.48 0.90(12) 38.88 0.82(12) 47.83 1.04(12)" 42.30 1.36(12) 41.10 0.65(11) 40.55 1.40(10) 39.75 1.34(10) 36.58 0.92(12) differs significantly from control t-test a p<0.001, b p<0.05 66 Figure 14. The change ( r e l a t i v e to con t r o l = 100) of hematocrit and muscle water i n sockeye salmon during sea-water adaptation. The f i s h had previously been exposed to 0.65 mg/L DHA for 120-h i n fresh water. Points f o r hematocrit are mean % based on E x p t s . 1 , 2 and 3. Points f o r muscle water are mean % based on Expts. 2 and 3. The l e v e l of s t a t i s t i c a l s i g n i f i c a n c e of the change i s given at the base of each graph. Table XII. Percentage water i n gut of sockeye salmon a f t e r a 5-day exposure to 0.65 mg/L DHA i n fresh water followed by exposure to sea water (Expt. 2). Percentage water i n gut Time i n sea water Control Exposed hours Mean + SE(n) Mean + SE(n) 0 76.71 0.97(10) 77.86- 0.74(11) 24 76.11 0.52( 9) 80.24 a 1.71('9) 48 76.96 1.20( 6) 75.61 0.96(10) 72 77.60 0.70( 8) 79.77 1.33( 7) 96 76.88 0.65(10) 77.35 0.39( 8) 120 77.68 0.52( 8) 80.18 a 0.57( 8) d i f f e r s s i g n i f i c a n t l y from co n t r o l p<0.01 t - t e s t 68 Figure 14 i n d i c a t e s that the blood hematocrit was c o n s i s t e n t l y elevated at the end of the freshwater exposure phase (p<0.001) but dropped r a p i d l y to con t r o l values a f t e r the f i s h had been i n sea water f o r 24 h. Subsequently hematocrit remained close to or s l i g h t l y below co n t r o l values, although a f t e r 120 h i n sea water, the hematocrits of the exposed group again became lower than i n the controls (p<0.05). P a r a l l e l muscle water determinations (Table XIII) confirmed previous r e s u l t s that the DHA exposure led to a hydration i n fresh water; t h i s was shown to be followed by a dehydration a f t e r the f i s h had been i n sea water for 24 h (Fig. 14). By 48 h, however, the muscle water l e v e l s were restored to the co n t r o l range although some f l u c t u a t i o n was evident. In Expts. 2 and 3 complete muscle water regulation was regained by 72 h as i l l u s t r a t e d i n Fig.14. Measurements of the % water i n the gut during Expt.2(Table XII)showed that muscle dehydration (which approached but d i d not meet s t a t i s t i c a l s i g n i f i c a n c e i n Expt. 2) was accompanied by an increase i n gut water content at 24 h. The amount of water i n the gut was again s i g n i f i c a n t l y increased (p<0.05) a f t e r 120 h i n sea water although the muscle water content was by now back to con t r o l l e v e l s . Blood hematocrit, however was s i g n i f i c a n t l y reduced i n exposed f i s h of Expt. 2 at t h i s time (Table XI). During Expt. 1 one f i s h was sampled when, having l o s t equilibrium, i t appeared to be having serious problems with muscular coordination. These observations were made a f t e r the f i s h had been i n sea water f o r ^  70 h. At the 72 h sampling period, t h i s f i s h was s t i l l breathing r e g u l a r l y but was quite r i g i d . The r e s u l t s of plasma e l e c t r o l y t e a nalysis f o r t h i s Table XIII. Percentage water i n muscle of sockeye salmon a f t e r a 5-day exposure to 0.65 mg/L DHA i n fresh water followed by exposure to sea water (Expts. 2 and 3). Percentage water i n muscle Time i n sea water Control Exposed hours Mean SE(n) Mean SE(n) Experiment 2 0 24 48 72 96 120 74.79 0.42(10) 73.27 0.30( 9) 73.39 0.76( 6) 74.91 0.16( 8) 74.99 0.44(10) 74.92 0.23( 8) 75.69 0.18(11) 72.39 0.30( 9) 74.52 0.20(10) 74.51 0.24( 7) 74.46 0.19( 8) 74.99 0.27( 8) Experiment 3 0 79.50 0.53(11) 81.17 0.59(12) a 24 76.22 0.32(11) 75.34 0.43(12) 48 76.86 0.20(12) 76.17 0.78(10) 72 77.21 0.27(11) 77.38 0.49(10) 96 77.18 0.57(12) 77.18 0.31(10) 120 77.35 0.63(12) 77.74 0.22(12) a D i f f e r s s i g n i f i c a n t l y from c o n t r o l p<0.05. 70 moribund f i s h are compared i n Table XIV to mean values for controls and exposed groups taken at the 72 h sampling period and c l e a r l y i n d i c a t e that t h i s f i s h had l o s t the a b i l i t y to regulate plasma i o n i c l e v e l s . The r e l a t i o n of behavioral symptoms to hydromineral disturbance was further investigated during Expts. 2 and 3. Moribund and D r i f t i n g F i s h As the analysis of plasma i o n i c composition was not conducted u n t i l a f t e r the completion of both Expts. 1 and 2, the presence or extent of i o n i c disturbance was not known during the second experiment. Based on behavioral observations, however 15 moribund f i s h were c o l l e c t e d during the course of Expt. 2 at various stages of d e b i l i t y and muscle and gut water determinations were conducted. These r e s u l t s together with a d e s c r i p t i o n of symptoms are presented i n Table XV and i l l u s t r a t e d i n F i g . 15 f o r comparison with exposed and co n t r o l groups i n Expt. 2. The r e s u l t s i l l u s t r a t e d i n Figure 15 i n d i c a t e that the muscle water content during the freshwater exposure period was greatly elevated i n d i c a t i n g hydration which was subsequently followed by a t i s s u e dehydration whose sev e r i t y appeared to be r e l a t e d to the time of seawater residence. P a r a l l e l measurements revealed a concurrent r i s e i n the amount of water i n the stomach, although i t appears that t h i s water ingestion may have been e l i c i t e d i n some f i s h several hours p r i o r to seawater entry (Fig. 15). Muscle water l e v e l s (Table XV) i n "inverted" f i s h ( f i s h which had l o s t equilibrium) suggest that the de-hydration may already be advanced at t h i s point i n the i n t o x i c a t i o n sequence. The presence of much higher water content i n the gut i n one f i s h (3.0 h i n Table XV which was sampled immediately a f t e r equilibrium l o s s suggests that the water accumulation may have been triggered by a combination of Table XIV. The plasma i o n i c composition of an "exposed" f i s h which l o s t equilibrium a f t e r 72 h i n sea water compared to mean values for controls and the "normal" exposed f i s h sampled a f t e r 72 h i n sea water i n Expt. 1. Control Exposed Inverted Sum of ions mEq/L 304.4 307.1 387.6 Chloride mEq/L 133.2 130.8 177.5 Sodium mEq/L 159.4 164.7 187.3 Potassium mEq/L 3.95 3.41 4.47 Calcium mEq/L 5.88 5.93 7.33 Magnesium mEq/L 1.99 2.30 11.00 Hematocrit % 38.3 36.2 36.4 72 Table XV. Size, percentage muscle and gut water and description of symptoms i n f i s h which appeared to be seriously affected during the course of Expt. 2. Time hours Fork length cm Wet weight g Muscle Water % Gut Water % Observations Fresh water exposure Time in sea water 103. ,2 13. ,4 28. ,05 78. 66 72. ,78 MB , YF 103. .2 13. .5 28. ,80 78. 36 75. ,32 MB, YP rigid 115. .1 16. .1 48. ,47 77. 16 85. ,05 YF 115. .1 15. .5 36. ,42 77. ,97 81. ,56 Fungus on gii; 120. .0 15. .4 36. ,80 78. .14 85. .67 MB 2. .0 15. .2 35. .20 78. .34 89. ,67 MB 3. .0 15. .1 35. .20 76. .29 88. .05 IJ 15. .4 . 14. .5 33. .89 74. .01 89. .79 MB, SGG 15. .4 14. .4 31. .96 76. .77 87. . 39 MT, .' SG, HM 15. .4 15. .4 40. .68 74. .44 91. .22 SG, HM 15. .4 14. .9 35. .72 74. .13 87. .24 SG 19. .6 15, .3 33. .15 73, .61 90. .06 MB, , SGG, YP 20. .3 13. .2 23. .71 71. .22 90, .07 MT, , SGG3 24. .4 15, .2 36, .10 72, .91 80, .23 I, SG, YP 44. .6 15, .2 35. .79 72. .89 87, .71 I, SG, HM, YP 88. .2 14, .7 28, .80 72 .71 80 .38 MT lhr 1 HM hemorrhage in fin membranes I (J) inverted (just) MB moribund - s t i l l breathing MT collected after death SG swollen gut SGG greatly swollen gut YF yellow fish YP yellow plasma 2 Control fish 3Gut contents 780 mOsmAg (SW 824 mOsm/kg) 73 D) CD Ejrposure Time in sea water (hours) * P <0.05 Figure 15. Relations between muscle (a) and gut water % (b) determinations on moribund f i s h and mean values f o r exposed and cont r o l groups i n Expt. 2. V e r t i c a l bars indicate ±SE of the mean. 74 toxicant and seawater exposure, as the muscle water content of t h i s f i s h (76.29%) was s t i l l higher than that of the c o n t r o l (74.79%) or of the exposed (75.69%) groups (Table XIII). Among the symptoms l i s t e d i n Table XV i s that of a yellowish tinge of the f i s h which was accompanied by a yellowish plasma i n those cases i n which blood samples were taken. This c o l o r was l a t e r a t t r i b u t e d to a form of toxicant-induced jaundice and i s discussed i n Appendix I I I . As the observations made on moribund f i s h i n Expt. 2 suggested a serious osmotic imbalance; during the course o f Expt. 3 three f i s h were sampled at 48, 72 and 96 h when i t became apparent that they could no longer maintain p o s i t i o n against the current. These f i s h were termed " d r i f t e r s " and were analyzed separately i n the hope of e s t a b l i s h i n g a l i n k between t h e i r behavior and the degree of i o n i c imbalance. Based on past observations, " d r i f t i n g " f i s h generally expired within 24-48 h, so that they would have probably added to the o v e r a l l m o r t a l i t i e s by the conclusion of Expt. 3. The plasma e l e c t r o l y t e l e v e l s of d r i f t i n g f i s h i n Expt. 3 are compared to those of c o n t r o l and exposed groups i n Figs.16 A to F and show that a l l the ions measured were considerably above t h e i r respective "exposed" means. The breakdown of t o t a l ions (Fig.16 A) i n d i c a t e s that the greatest changes appear-••.i.> ed to have occurred i n plasma Mg + + (Fig.16 F) which seemed to be increasing with time i n sea water. Plasma C a + + regulation (Fig.16 E) i n exposed groups was restored within 48 h and there was some subsequent overcompensation when compared to c o n t r o l s . However, plasma C a + + l e v e l s i n d r i f t i n g f i s h remained roughly double at t h i s time. The major plasma cations Na + and C l 4 1 0 3 9 0 3 7 0 h 3 5 0 Total Ions CJ 0) 3 3 0 310 2 9 0 270 C o n t r o l E x p o s e d Drif ter » 1 0 1 1 24 4 8 i i 72 9 6 1 . 120 Time in sea water (hours) | 0.01 0.001 0.05 N S NS NS Figure 16, A comparison of plasma e l e c t r o l y t e concentrations (a-f) measured i n 3 d r i f t i n g f i s h , with the means of co n t r o l and exposed groups during Expt. 2. V e r t i c a l bars indicate ±SE of the mean and the l e v e l of s i g n i f i c a n c e of the dif f e r e n c e between control and exposed means i s given i n the box below each graph. Time in sea water (hours) 77 78 p e r s i s t e d at greatly elevated l e v e l s i n d r i f t i n g f i s h at a time when the previously exposed but be h a v i o r a l l y normal f i s h were regu l a t i n g these ions towards c o n t r o l values (Fig. 16 B,C). While plasma K + l e v e l s remained closer to "exposed" means than any of the other ions, they d i d appear to be increasing with time i n sea water. These experiments confirmed that the osmotic imbalance brought on by sublethal exposure of salmon to DHA i n fr e s h water was also accompanied by an e l e c t r o l y t e disturbance. Furthermore, the subsequent t r a n s f e r of these f i s h i n t o clean sea water r e s u l t e d i n a marked l o s s i n the p r e c i s i o n of plasma ion regulation and i n a general dehydration. As the maintenance of hydromineral balance i n salmonids r e l i e s h e avily on a reduction of the g i l l permeability and an increase i n the gut per-meability to water, an experiment was conducted to measure the e f f e c t s of DHA exposure on t h i s aspect of the function of these two osmoregulatory organs. A l t e r a t i o n s i n the permeability of e i t h e r organ could contribute to the ra p i d r i s e i n plasma e l e c t r o l y t e l e v e l s which was observed above. B. EFFECTS OF DHA ON GILL AND GUT PERMEABILITY TO WATER INTRODUCTION The movement of euryhaline f i s h i n t o sea water from the freshwater environment t r i g g e r s a complex sequence of adaptive mechanisms which act i n concert to maintain mineral homeostasis. Although r a p i d dehydration i s avoided through a reduction i n the permeability of the branchial epithelium to water (Maetz, 1970a) a net water l o s s s t i l l occurs and sea water i s swallowed, absorbed across the gut and subsequently p h y s i o l o g i c a l l y " d i s t i l l e d " to replace the free water l o s t by the g i l l s (Smith, 1930). The reduction i n g i l l permeability serves to l i m i t t h i s i n e v i t a b l e water loss while the mechanism of i n t e s t i n a l water transport enables the f i s h to replace branchial and renal water losses. Impediment i n the reduction of free water movement across the g i l l s would place an a d d i t i o n a l load on the gut to absorb more water and consequently on the branchial and renal ion pumps which dispose of the excess s a l t . Since the e l e c t r o l y t e imbalance observed during the present study could have or i g i n a t e d i n a t o x i c a n t - r e l a t e d interference with normal per-meability changes i n the g i l l and/or gut, an experiment was conducted to t e s t whether sublethal exposure of sockeye salmon to DHA i n fresh water i n t e r f e r e d with subsequent permeability changes i n the g i l l s and i n t e s t i n e s during sea water adaptation. The water permeability of the i s o l a t e d g i l l and the water transport a b i l i t y of the i s o l a t e d gut were measured i n DHA-exposed f i s h a f t e r a 24 h recovery period i n sea water. Based on the i s o l a t e d g i l l technique of Bellamy (1961) and Utida et a l . (1967), a procedure was developed to measure the change i n buoyancy of an i s o l a t e d g i l l arch incubated i n sea water f o r 1 h. This change i n buoyancy 80 was assumed to be due to the loss of water across the g i l l epithelium making the preparation more dense and therefore sinking deeper i n the sea water and r e g i s t e r i n g an apparent weight increase on an electrobalance. The water transport a b i l i t y of the gut was measured by the weight changes of i n t e s t i n a l sacs (Oide and Utida, 1967; Utida et a l . , 1967) f i l l e d with and incubated i n Cortland s a l i n e (Wolf, 1963). To enable sampling of i n d i v i d u a l f i s h without disturbance of the others, an apparatus was designed i n which electroshock was used to ra p i d l y stun a selected i n d i v i d u a l with no disturbance of the other f i s h i n the tank. MATERIALS AND METHODS Two s i m i l a r experiments were conducted, one i n June and the second i n July 1978. In both experiments, ten f i s h were exposed to 0.4 mg/L DHA i n fresh water f o r 120 h. An equal number of f i s h served as controls. The toxicant was then discontinued, FW replaced by SW, and 24 h l a t e r i n d i v i d u a l f i s h were removed and the g i l l and gut prepared f o r incubation. The exposure to 0.65 mg/L DHA which was sublethal to sockeye salmon during the i o n i c balance experiments proved to be l e t h a l to f i s h confined i n the tubes used f o r the permeability study. The choice of 0.4 mg/L f o r the g i l l permeability experiments was made on the basis of a comparison of the time to 50% mo r t a l i t y (LT50) of the "tubed" f i s h exposed to 0.65 mg/L DHA,to the t o x i c i t y curve (Fig. 9) obtained previously f o r free-swimming f i s h . By extrapolation, 0.4 mg/L DHA was chosen to ensure 100% f i s h s u r v i v a l during a 5-day exposure period. Sockeye salmon smolts were obtained from the Great Central Lake run i n March 1978 and kept i n outdoor holding tanks at PEI under conditions described i n the General Methods section. Approximately 25 f i s h were dip netted from the outdoor tanks and transferred to the laboratory donut tanks i n a bucket 81 containing 33 mg/L MS222 dissolv e d i n water of 10 /oo s a l i n i t y . F i s h were given a minimum of 96 h to acclimate to the flow conditions i n the laboratory, were fed once d a i l y with OMP, and starved f o r 24 h p r i o r to tr a n s f e r to the experimental tanks. At the time of tr a n s f e r , a l i g h t anesthesia was brought on by the addition of a seawater s o l u t i o n of 2 g MS222 to the tank (36 mg/L) a f t e r shutting o f f the water supply and adding an air-stone l i g h t l y bubbling oxygen in t o the water. As soon as the f i s h began to d r i f t with the current, they were netted and tra n s f e r r e d to the tubes i n the experimental tanks. F i s h of comparable size were selected and d i s t r i b u t e d a l t e r n a t e l y between the contr o l and t e s t tanks. Each tank contained 10 f i s h which were placed i n i n d i v i d u a l p l a s t i c tubes suspended i n the water current (Fig.17 ). The up-stream end of each tube had a 6 mm mesh netting glued to i t , while at the downstream end, the netting was attached to a removable s p l i t p l a s t i c r i n g . A f t e r the f i s h was inserted, the r i n g was replaced and following recovery from anesthesia, the f i s h rested q u i e t l y on the tube bottom or swam slowly against the current. Attached to the length of each tube were two s t r i p s of 2.5 cm aluminum tape (3M Scotch No. 425) forming electrodes on ei t h e r side of the f i s h (Fig.18 ). Each tube was wired to a Variac through a switching c i r c u i t so that voltage could be applied to one tube at a time. Each tube was also covered with black p l a s t i c to minimize v i s u a l disturbance. Using t h i s apparatus, i n d i v i d u a l f i s h could be instantaneously stunned i n t h e i r tubes and removed from the water without d i s t u r b i n g the upstream f i s h . The f i s h were given an acclimation period of 72 h before the s t a r t of the toxicant exposure. An airi-stone ensured that diss o l v e d oxygen l e v e l s remained i n excess of 90% saturation throughout the experiments and the temperature was maintained at 11.5 ±0.S^C.After the 120 h exposure period, followed by 24 h 82 Figure 17. I l l u s t r a t i o n of a section of a donut tank showing the p o s i t i o n i n g of p l a s t i c tubes to which sockeye salmon were confined during a 120 h exposure to 0.4 mg/L DHA i n fresh water. Figure 18. Detailed i l l u s t r a t i o n of one of the p l a s t i c tubes used for the containment of sockeye salmon during t h e i r exposure to 0.4 mg/L DHA p r i o r to the g i l l permeability experiments. 84 i n sea water, i n d i v i d u a l f i s h were stunned by electroshock (20V AC f o r 10s.). The tube containing the immobilized f i s h was c a r e f u l l y l i f t e d from the water so as not to dis t u r b the neighboring f i s h . The f i s h was then removed from the tube, stunned by a blow on the head, measured, b l o t t e d and weighed. The e n t i r e branchial basket was then r a p i d l y removed as shown i n Fig. 19, 1-12 with frequent r i n s i n g with c h i l l e d (10°C) Cortland s a l i n e . During step 3, bleeding from the severed pseudobranchial artery was checked by means of cautery. A f t e r removal, the f i r s t b r a nchial arch was t i e d o f f with s i l k and separated from the r e s t of the g i l l bars of the branchial basket. A s t a i n l e s s s t e e l hook (0.46 mm x 1.5 cm) was glued to the v e n t r a l i n s i d e surface of the f i r s t g i l l bar with a drop of Eastman 910 cyanoacrylate adhesive. A f t e r a r i n s e with deionized water, the e n t i r e preparation was transferred to the beaker of the g i l l incubation apparatus (Fig.20 ) i n which water from the constant temperature bath was c i r c u l a t e d through the beaker water jacket. The beaker was f i l l e d with aerated sea water (810 mOsM) and temperature e q u i l -i b r a t e d while the d i s s e c t i o n s were c a r r i e d out. The g i l l preparation was then o -incubated for 1 h at 11.9 +0.01 C (X ±SE) . The apparent change i n g i l l weight was monitored on a Sanyo 4092U Video Monitor using a Sanyo Video Camera VC1150 and recorded on a Sanyo Video Tape Recorder (VTR 1200) f o r future processing. At the end of the g i l l incubation period, the preparation was removed from the balance, the g i l l bars dissected away from the c e n t r a l c a r t i l a g e (Fig. 19, 13-14) placed i n tared aluminum dishes and oven dri e d to constant weight. The weight change of the g i l l was expressed i n mg/100 mg dry g i l l weight/hr. The gut ex t r a c t i o n was started as soon as the g i l l incubation was under way (^  5 min from f i s h electroshock). The abdominal c a v i t y was opened and Dissection procedure followed to obtain the filament preparation used i n the g i l l permeability experiment. Figure -12 shows the f i n i s h e d f i r s t branchial arch u n i t . A f t e r completion of the experiment, the c a r t i l a g e was trimmed o f f (Figure -13 and -14) p r i o r to the determination of dry weight. Figure 20- The apparatus used to measure the weight loss of i s o l a t e d sockeye salmon g i l l filaments incubated i n sea water. The f i s h had been previously exposed to 0.4 mg/L DHA f o r 120 h i n fresh-water. oo 87 the i n t e s t i n e was transected i n the region of the vent, freed from the supporting mesentery a n t e r i o r l y and cut j u s t p o s t e r i o r to the l a s t p y l o r i c o caecae. The gut was flushed clean with c h i l l e d (10 C), aerated Cortland s a l i n e (285 mOsM), then f i l l e d with 200 u l sal i n e by means of a 1 ml syringe f i t t e d with a 21 G needle tipped with 1 cm of PE 60 (Intramedic) polyethylene tubing. Both ends of the i n t e s t i n a l sac were t i e d o f f with s u r g i c a l s i l k , adhering f a t was gently teased o f f , and a f t e r b l o t t i n g , the preparation was weighed to the nearest mg on a Mettler H 43 a n a l y t i c a l balance. I t was then transferred to the incubation bath (Fig. 21) containing l i g h t l y aerated Cortland s a l i n e at 11.9 ±0.1°C (X ±SE) for 1 h. At i n t e r v a l s of approximately 10 min, the sac was removed, b l o t t e d , weighed, arid replaced i n the s a l i n e . A f t e r the f i n a l weighing, the s i l k was snipped o f f , and the gut was d r i e d to constant weight i n a tared aluminum dish i n a forced d r a f t oven at 105°C. Weight change was expressed i n mg/100 mg dry gut wt./h. RESULTS As the changes i n weight of co n t r o l g i l l s i n Expts. 1 and 2 were not s i g n i f i c a n t l y d i f f e r e n t , t h e data were pooled (Table XVI). The combined r e s u l t s i n d i c a t e that the p r i o r sublethal exposure of sockeye salmon to DHA i n f r e s h water l e d to a s i g n i f i c a n t l y greater weight los s by t h e i r g i l l s i n sea water. The r e s u l t s of the gut incubation experiment ind i c a t e that while the amount of water transported by the i n t e s t i n e s of previously exposed f i s h was considerably lower than the corresponding controls (Table XVII), the amount of v a r i a t i o n i n both groups was large and the di f f e r e n c e was not s i g n i f i c a n t . Figure 21. Diagram showing the apparatus used f o r the incubation of i s o l a t e d i n t e s t i n a l sacs taken from sockeye salmon which had previously been exposed to 0.4 mg/L DHA for 120 h. Table XVI. F i s h s i z e and the loss i n weight (during sea water incubation)of g i l l arches i s o l a t e d from juv e n i l e sockeye salmon which had been previously exposed to 0.4 mg/L DHA i n fresh water f o r 5 days* 3. (Mean ±SE) Fork length cm Wet Weight g G i l l weight l o s s mg/100 mg dry weight/h Control (19) 14.3 ±0.16 30.48 ±0.87 5.685 ±0.27 Exposed (17) 14.7 ±0.18 31.99 ±1.00 6.471 ±0.25 aWeight of water l o s t . G i l l s were extracted from f i s h which had been i n SW f o r 24 to 36 h. °Differs s i g n i f i c a n t l y from c o n t r o l p<0.05 ( t - t e s t ) . Table XVII. F i s h s i z e and the loss i n weight of i s o l a t e d i n t e s t i n a l sacs taken from j u v e n i l e sockeye salmon which had previously been exposed to 0.4 mg/L DHA i n fresh water f o r 5 d a y s . a ' b (Mean ±SE) Fork length Wet Weight cm g Gut weight l o s s mg/100 mg dry weight/h Control (14) 14.1 ±0.17 29.44 ±0.87 45.18 ±4.37 Exposed (17) 14.7 ±0.18 31.99 ±1.00 37.73 ±4.31 Intestines were extracted from f i s h which had been i n SW f o r 24 to 36 h. I n t e s t i n a l sacs f i l l e d with and incubated i n Cortland s a l i n e . 91 DISCUSSION Water and e l e c t r o l y t e homeostasis i n f i s h i s achieved through the coordi-nated functions of the g i l l , gut and kidney. Since plasma e l e c t r o l y t e l e v e l s normally are maintained within a r e l a t i v e l y narrow range,their measurement provides a broad assessment of the performance of t h i s integrated homeostatic function. General deviations from the normal range do not explain which cont r o l mechanisms have been disturbed but the patterns of osmotic and i o n i c change i n r e l a t i o n to the external environment can suggest which mechanisms may be involved. However,the s p e c i f i c mechanisms by which the toxicant (DHA) a f f e c t s hydromineral balance were not studied i n the present i n v e s t i g a t i o n . The present study established that sublethal exposure to DHA a l t e r e d osmotic and i o n i c balance i n sockeye salmon smolts i n fresh water. In addition, when previously exposed f i s h were tr a n s f e r r e d into sea water, hydro-mineral balance was again disturbed f o r several days before returning to normal. Exposure to DHA also l e d to a change i n g i l l permeability. An observed accumulation of f l u i d i n the gut could not be a t t r i b u t e d to a d i f f e r e n c e i n the water absorptive function of the i n t e s t i n e but could represent an increase i n the ingestion of sea water i n response to dehy-dation i n the hypertonic medium. Studies on DHA uptake showed that the r e s i n a c i d accumulates several-hundred-fold i n the t i s s u e s , notably the g i l l and the kidney; which are organs d i r e c t l y involved i n e l e c t r o l y t e balance i n salmon. A consideration of the o v e r a l l changes observed i n hydromineral balance and i n several hematological parameters suggests three p o s s i b l e patterns of t o x i c a c t i o n : 1) DHA d i r e c t l y a f f e c t s g i l l and gut permeability and increases the passive movements of water and e l e c t r o l y t e s . 2) DHA exposure 92 leads to hypoxia and the observed changes i n hematocrit and e l e c t r o l y t e s represent a secondary response; 3) DHA a f f e c t s kidney function. Although i t i s of course p o s s i b l e that a l l three of these actions occur simultaneously, they nevertheless have been p a r t i t i o n e d f o r the purposes of the discussion and as a r e s u l t some r e p e t i t i o n has been unavoidable. Changes i n Permeability - G i l l Among the mechanisms u t i l i z e d by euryhaline f i s h to maintain osmotic and i o n i c balance i n media d i f f e r i n g widely i n osmotic pressure and i o n i c compo-s i t i o n i s that of a change i n g i l l permeability. The maintenance of low osmotic permeability of the g i l l i s e s s e n t i a l to minimize water gain i n fr e s h water and thus reduces the amount of work required by the kidney.to maintain water balance. When euryhaline f i s h move from f r e s h water i n t o the marine environment, a reduction i n g i l l permeability functions to diminish the osmotic l o s s of water due to the gradient between the blood plasma (^  300 mOsM/kg) and sea water <> 1000 mOsM/kg). Evans (1969) reported a lower g i l l water permeability i n SW-adapted as compared to FW-adapted flounder and e e l s . However, there e x i s t s a c e r t a i n time lag before these adaptive permeability changes are complete. The rate of bra n c h i a l water l o s s i n eels j u s t a f t e r t r a n s f e r from FW to SW was almost t r i p l e that observed a f t e r 3 days i n SW (Oide and Utida, 1968) and Kamiya (1967) found that g i l l s excised from eels a f t e r 6-12 h adaptation to SW l o s t s i g n i f i c a n t l y l e s s water than FW g i l l s and i f the f i s h were given 24 h i n SW, t h e i r g i l l s behaved as SW g i l l s . Because of t h i s time lag i n the establishment of g i l l permeability c h a r a c t e r i s t i c s , i n the present study sockeye salmon were given at l e a s t 24 h of sea water adaptation p r i o r to g i l l and gut ex t r a c t i o n . The period i n sea water varied from 24 ^ 36 h as i t took about 75 min f o r a complete experiment to be performed on one f i s h . 93 A f t e r entry of the f i s h i n t o the sea, osmotic loss of water i s recovered by ingestion of SW which i s then absorbed across the i n t e s t i n e and subsequently p h y s i o l o g i c a l l y " d i s t i l l e d " with the bulk of Na + and C l excretion occurring across the g i l l s . As these mechanisms take some time to become f u l l y oper-a t i o n a l (Kirsch and Mayer-Gostsn, 1973), the reduction i n g i l l permeability may be an e f f e c t i v e and e n e r g e t i c a l l y inexpensive method of l i m i t i n g dehydration upon entry into sea water. The strategy used to accomplish adaptation to sea water appears to vary with species. Lahlou et a i . (1975) showed that the trout Salmo i r r i d e u s depends p r i m a r i l y on reduction of peri p h e r a l water l o s s and therefore l e s s on the active uptake of ions and water i n the i n t e s t i n e . This e n e r g e t i c a l l y economical strategy was li n k e d to the observation of a r e l a t i v e l y slow start-up time for the g i l l Na + excretory mechanism. In t h i s case, l i m i t i n g the loss of water appears to be "cheaper" than replacing i t a f t e r l o s s . The maintenance of an e s s e n t i a l l y homoiosmotic i n t e r n a l concen-t r a t i o n during adaptation to sea water i n the rainbow trout r e l i e s heavily on a rapid reduction of osmotic permeability (Gordon, 1963), much f a s t e r than some other euryhaline species such as the e e l , flounder (Motais et a l . , 1966) and the k i l l i f i s h (Maetz et a l . , 1967). In the present study, normal sockeye salmon were observed to maintain e s s e n t i a l l y homoiosmotic regulation during adaptation to sea water. On the other hand the salmon which had been pre-exposed to DHA i n fresh water showed a considerable los s of plasma e l e c t r o l y t e regulatory p r e c i s i o n a f t e r t r a n s f e r to sea water. I f sockeye salmon r e l y on a reduction i n permeability to a s i m i l a r extent as do tr o u t , the observed i o n i c and osmotic disturbance may be li n k e d i n part to a toxicant-induced increase i n g i l l permeability. The more pronounced water l o s s across i s o l a t e d g i l l s of salmon which had been previously exposed to DHA supports t h i s hypothesis. 94 In many species, during the time that osmotic permeahility i s being reduced, there i s a progressive augmentation of branchial permeability to Na + and C l (Maetz, 1974) as well as an increase i n the i n t e s t i n a l s a l t pumping e f f i c i e n c y and i t s attendant s o l u t e - l i n k e d water flow (Skadhauge, 1969). In three euryhaline species (the flounder, e e l and k i l l i f i s h ) the very low rate of sodium turnover i n FW (0.01 to 0.55% i n t e r n a l Na +/h)rose to 29.5 to 46.9% i n sea water (Maetz, 1970b), with the rate of turnover generally d i r e c t l y p r oportional to s a l i n i t y . These species r e l y on the r a p i d a c t i v a t i o n of a powerful s a l t pumping mechanism i n the g i l l s and the i n t e s t i n e to maintain s a l t and water balance; accordingly, t h e i r permeability to both s a l t and water i s high and during adaptation to sea water, the e l e c t r o l y t e l e v e l s i n the blood plasma depart considerably from FW l e v e l s . In marked contrast, the rainbow tro u t maintains a low Na + turnover i n sea water (4-10%/h) as well as an only s l i g h t l y increased C l turnover rate (Lahlou e_t al^. , 1975; Gordon, 1963). Lahlou et a l . (1975) also found that when trout were m i l d l y stressed -during seawater adaptation, Na + i n f l u x increased by 4-5 times while outflux only doubled, leading to a net gain of sodium u n t i l death. In the trout, the strategy appears to be p r i m a r i l y one of l i m i t i n g s a l t loading and water l o s s during adaptation to sea water. Thus although e n e r g e t i c a l l y advantageous, such a strategy w i l l render species using i t more vulnerable to hydromineral disturbance by f a c t o r s which d i r e c t l y or i n d i r e c t l y lead to increased g i l l permeability, as the homeostatic mechanisms which excrete s a l t s and absorb water may be e a s i l y overloaded. I f the strategy followed by the rainbow trout i s representative of salmonids i n general, then a r a p i d s a l t loading of DHA-exposed sockeye salmon may occur, p l a c i n g an excessive burden on osmoregulatory mechanisms which are 95 designed to avoid s a l t loading rather than to deal with i t . Although the permeability of the g i l l s to s a l t was not determined i n t h i s study, an augmentation i n branchial i o n i c permeability could explain the rapid r i s e i n plasma e l e c t r o l y t e l e v e l s i n DHA-exposed salmon undergoing adaptation to sea water. The mechanisms behind the apparent increase i n g i l l permeability induced by DHA exposure are unknown. Residue analysis showed that DHA was accumulated by the g i l l s (Appendix 1-4) so a d i r e c t action on the permeability c h a r a c t e r i s t i c s of the branchial epithelium may have occurred. Another p o s s i b i l i t y could be that i n response to DHA-induced hypoxia a greater e f f e c t i v e exchange area was perfused with blood at the time that the g i l l s were extracted and incubation of g i l l arches i n sea water led to a more rap i d water l o s s . Increases i n the f u n c t i o n a l surface area of the g i l l s have been shown to occur i n response to hypoxic stress (Steen and Kruysse, 1964; Holeton and Randall, 1967a) and can r e s u l t i n an increase i n passive water movements (Loretz, 1979). Both of these phenomena would be expected to render l e s s e f f e c t i v e the reduction i n branchial permeability necessary f o r successful seawater adaptation. The e f f e c t of such a dysfunction would be to increase water l o s s and s a l t gain and may have contributed to the e l e v a t i o n i n plasma e l e c t r o l y t e l e v e l s of sockeye salmon observed i n the sea water stage of the present study. The time course of recovery of hydromineral balance as ind i c a t e d by the gradual return of plasma e l e c t r o l y t e s to c o n t r o l values suggests that g i l l permeability may be disturbed f o r ^ 3 days a f t e r sublethal DHA exposure. A l l plasma e l e c t r o l y t e and muscle water l e v e l s had returned to normal by 120 h i n sea water i n d i c a t i n g that i r r e v e r s i b l e changes had not occurred. 96 The presence of calcium ions appears to be e s s e n t i a l i n f i s h e s to ensure successful adaptive permeability changes to both water and e l e c t r o l y t e s CPotts and Fleming, 1970, 1971). This e f f e c t may be l a r g e l y a passive process r e l a t e d to changes i n the s t r u c t u r a l properties of the d i f f u s i o n b a r r i e r of the g i l l s , such as s t a b i l i z a t i o n of the mucus coat or the c e l l membrane (Mashiko and Jozuka, 1962). The addition of calcium to fresh water i n amounts equivalent to seawater concentration reduced the urine flow i n the brown trou t (Salmo trutta) by 32% (Oduleye, 1975). As urine flow i s a measure of the osmotic permeability i n freshwater f i s h , t h i s reduction i n flow may represent a s i g n i f i c a n t decrease i n the work required of the kidney. The observed elevation of plasma C a + + l e v e l s may be an adaptive response i n an e f f o r t to reduce the permeability of the g i l l s to water i n f l u x . As the calcium content of the fresh water used i n the present study was very low (0.8 mEq/L) i t i s doubtful that the r i s e i n plasma C a + + was due to an increased uptake. More l i k e l y i s the m o b i l i z a t i o n of calcium from r e s e r v o i r s such as scales and bone (Mashiko and Jozuka, 1962) which can serve as a calcium source when required during times of stress (Ichikawa, 1953; U r i s t , 1966; Weiss and Watabe, 1978). Although the reduction i n g i l l permeability by the presence of external calcium has been well established, I was not able to f i n d any l i t e r a t u r e on whether an e l e v a t i o n of plasma C a + + l e v e l s from within has a s i m i l a r e f f e c t . Plasma C a + + l e v e l s i n freshwater f i s h are known to be maintained by p r o l a c t i n secretion, which i n turn i s c o n t r o l l e d by environmental Ca and Mg concentrations. Ogawa (1974) showed that p r o l a c t i n i n j e c t i o n s i n t o e e l s and rainbow tro u t p r i o r to s a c r i f i c e reduced the water permeability of i s o l a t e d g i l l s incubated i n deionized water. I t appears probable that a lowering of plasma Ca l e v e l s may t r i g g e r p r o l a c t i n s e c r e t i o n leading to an 97 increase i n plasma Ca concentration and p o s s i b l y i t s binding at the g i l l s which then influences plasma i o n i c composition and osmolality (Wendelaar Bonga, 1978). Changes i n Permeability - Gut The a b i l i t y of the i s o l a t e d i n t e s t i n e to transport water appeared to be l i t t l e a f fected i n f i s h exposed to DHA i n fresh water and then t r a n s f e r r e d to sea water f o r 24 h. However, the increased water content i n the stomachs of salmon which were dehydrated may i n d i c a t e that the rate of water absorption across the gut could not keep up with the rate of water los s across the g i l l s i n these f i s h . Under normal circumstances, the process of seawater absorp-t i o n involves an i n i t i a l movement of plasma water i n t o the gut (Utida e t a l . , 1967; Skadhauge, 1969); excessive ingestion may lead to a further dehydration and s a l t loading of plasma (Kirsch and Mayer-Gostan, 1973). Dehydrated f i s h cannot gain more water by drinking more as there i s a l i m i t to the amount of s a l t and i t s attendant water flow that the i n t e s t i n e i s capable of absorbing, e s p e c i a l l y during the e a r l y stages of adaptation to sea water (Skadhauge, 19691. This emphasizes the importance of reducing osmotic permeability of the g i l l s during sea water adaptation (Kristensen and Skadhauge, 1974). Although the ingestion of some fresh water by t e l e o s t s has been well documented, (Maetz and Skadhauge, 1968; G a i t s k e l l and Chester-Jones, 1971; Potts and Fleming, 1970) and i s not considered to be e n e r g e t i c a l l y d i s -advantageous (Shuttleworth and Freeman, 1974), the ingestion of abnormal amounts of f r e s h water d i d lead to a r a p i d general hydration i n e e l s (Kirsch and Mayer-Goston, 1973).. In the present experiment, the increased ingestion of water was frequently observed during freshwater exposure of salmon to DHA. The d i s s e c t i o n of f i s h having a bloated appearance revealed a t u r g i d stomach f i l l e d with water. As the maintenance of water permeability, i n i t i a t i o n of 98 b r a n c h i a l s a l t excretion, and the drinking r e f l e x have been shown to be under the c o n t r o l of the nervous system (Mayer-Gostan and Hirano, 1976), the indicated s t i m u l a t i o n of drinking i n freshwater-adapted sockeye salmon may be r e l a t e d to the accumulation of high concentrations of DHA i n the b r a i n or other p a r t s of the nervous system. On a wet weight b a s i s , the b r a i n t i s s u e contained the highest l e v e l s of DHA of a l l the organs analyzed i n t h i s study (Appendix 1-4). In a d d i t i o n , the stomach t u r g i d i t y frequently observed during DHA exposure could be expected to exert considerable pressure on i n t e r n a l organs. Such a mechanical blockage could have i n t e r f e r e d with the normal flow of b i l e and contributed to a form of o b s t r u c t i v e jaundice observed i n DHA-exposed f i s h and which i s described i n Appendix I I I . Blood Hematocrit and Plasma E l e c t r o l y t e Changes The blood sampled from salmon exposed s u b l e t h a l l y to DHA i n f r e s h water frequently appeared to be more viscous and darker i n color than that taken from c o n t r o l f i s h , i n d i c a t i n g that some form of hemoconcentration had occurred. Hemoconcentration was further i n d i c a t e d by the highly s i g n i f i c a n t increase i n blood hematocrit observed i n f i s h a f t e r 120 h o f DHA exposure. On the other hand, the concurrent reduction i n plasma osmolality suggested that hemodilution had occurred, as might be expected i n f i s h s u f f e r i n g from an osmotic imbalance i n f r e s h water. A p o s s i b l e explanation of t h i s apparent discrepancy i s o f f e r e d a f t e r a discussion of three mechanisms which can act to increase hematocrit i n f i s h . An increase of hematocrit i s a well-known response of f i s h to hypoxia (Doudoroff and Shumway, 1970) which can be an adaptive measure to increase the oxygen carrying capacity of the blood. At l e a s t three mechanisms can contribute to a r i s e i n hematocrit: 1) a decrease i n plasma volume, 99 2) an act u a l increase i n red blood c e l l numbers (polycythemia) 3) an increase i n red c e l l s i z e due to swelling. Any one or combination of these changes w i l l lead to an ele v a t i o n i n hematocrit. A decrease i n plasma volume i n response to hypoxia can r e s u l t from movement of water from the blood to t i s s u e s (Hall, 1928; Black e t a l . , 1959; Soivi o et a l . , 1974c) or an increased d i u r e s i s (Westfall, 1943; Hunn, 1969; Swift and Lloyd, 1974; Lloyd and Swift, 1976). These f l u i d s h i f t s can r e s u l t i n hemoconcentration and a r i s e i n hematocrit. In the present study, nemo-concentration due to f l u i d s h i f t s from the plasma i n t o the tissues can be ru l e d out since the plasma osmolality i n DHA exposed f i s h i n fresh water dropped (Fig. 12). rather than increased as might be expected i f plasma water were being removed from the c i r c u l a t i o n . A f t e r t r a n s f e r to sea water, plasma osmolality was s i g n i f i c a n t l y elevated at 24 h but hematocrit was not. However, when plasma osmolality of both groups became e s s e n t i a l l y i d e n t i c a l at 120 h, the hematocrits of exposed f i s h were s i g n i f i c a n t l y below those of controls. An actual increase i n the numbers of red blood c e l l s can r e s u l t from a stimulation of hematopoietic a c t i v i t y and/or release of erythrocytes from storage depots such as the head kidney (Ostroumova, 1957; Zanjani et a l . , 1969; Fromm, 19-77).. Some authors have suggested that the spleen may also serve as a storage organ f o r erythrocytes which can be released i n t o the c i r c u l a t i o n i n response to hypoxic st r e s s (Dawson, 1935; Lloyd and Swift, 1976). I t i s doubtful however, that s p l e n i c contraction alone could contribute enough red blood c e l l s to a l t e r hematocrit s i g n i f i c a n t l y as Stevens (1968) determined the blood volume of the spleen i n rainbow trou t to be 70 yl/100 g body weight. Considering a blood volume of 5% of the body weight (Smith, 1966) and an 100 i n i t i a l hematocrit of 30%, even a complete emptying of the splenic contents (assuming i t s e n t i r e blood volume comprised erythrocytes) would only r a i s e the hematocrit to 31.3% i n a 100 g trout. I t i s more l i k e l y then that the spleen i s involved i n the uptake and destruction of red blood c e l l s and functions as a storage s i t e f o r i r o n i n the form of hemosiderin bodies (Grover, 1968; Yu et a l . , 1971) and responds to hypoxia by supplying the i r o n required f o r hemoglobin synthesis i n the hematopoietic t i s s u e of the kidney (Ostroumova, 1957). The evidence suggests that the contribution of the spleen to r a i s i n g hematocrit i s probably i n d i r e c t and not immediate. I f as suggested, DHA exposure i n f r e s h water l e d to hypoxia, a stimulation of erythropoiesis (red blood c e l l formation) may have occurred with red blood c e l l s being added to the c i r c u l a t i o n i n s u f f i c i e n t numbers to account f o r the higher hematocrit. Although red blood c e l l s counts were not performed, i t i s u n l i k e l y that the observed r i s e i n hematocrit (from 34% to 45%, t y p i c a l ) could have been due to polycythemia because within 24 h of recovery i n sea water, the hematocrit of exposed and c o n t r o l f i s h was s i m i l a r . This would have required a r a p i d removal of a large number of red blood c e l l s from the c i r c u l a t i o n . Although such a function was implied f o r the l i v e r and the spleen by Swift and Lloyd (1974), based on blood volume determinations f o r these organs (Stevens, 1968) i t seems u n l i k e l y that such a storage of red blood c e l l s would occur. Soivio et a l . (1974a) could not f i n d any p a r t i c u l a r area i n the trout kidney s p e c i a l i z e d f o r blood storage nor any evidence for a release of erythrocytes from that organ during hypoxia. The r i s e i n hematocrit during toxicant exposure i n f r e s h water followed by a r a p i d drop a f t e r the f i s h were tr a n s f e r r e d to sea water, when considered together with changes i n plasma osmolality, rather suggests a swelling and 101 shrinking of erythrocytes. I f indeed DHA exposure contributed to a hypoxic condition i n sockeye salmon through some interference with gas exchange mechanisms, then a buildup of CO2 could occur i n the blood. In salmonids, the swelling of red blood c e l l s i n response to hypoxic stress can be caused by small increases i n plasma CO2 l e v e l s (Benditt et a l . , 1941 ; Irv i n g et a l . , 1941). C e l l u l a r swelling contributed to increased hematocrits i n hypoxic rainbow t r o u t (Holeton and Randall, 1967a) and Soivi o et a l . (1974b,c) confirmed that the swelling observed i n vivo i n t r o u t could also be demon-strated i n v i t r o at reduced C>2 l e v e l s . The incubation of swollen erythrocytes i n O2 d i d not r e s u l t i n complete volume r e g u l a t i o n and these authors concluded that the volume change was a complex response i n v o l v i n g changes.in pH, PCO2 and l a c t i c a c i d as well as some unknown metabolite. C a s i l l a s and Smith (1977) reported t h a t a l o c a l i z e d t i s s u e hypoxia a f t e r muscular exertion i n t r o u t re s u l t e d i n a swelling of erythrocytes and l e d to a sharp r i s e i n hematocrit. The in_ v i t r o a d d i t i o n of l a c t i c a c i d to the blood of the sucker (Catastomus  commersoni)led to a 30% increase i n hematocrit also as a r e s u l t of erythrocyte swelling (Black and Irvin g , 1938). Hypoxic conditions may contribute to a buildup of these metabolites i n the t i s s u e s or i n the red blood c e l l s per se. Eddy (1977). determined that the O2 uptake by the blood formed a s i g n i f i c a n t portion (4.5% at 20°C) of the t o t a l energy consumption of tro u t , and may be associated with the metabolic processes of the red blood c e l l . Recent work has shown that volume changes i n red blood c e l l s of f i s h can also be brought about by osmotic and i o n i c movements as part of the mechanism of "isosmotic i n t r a c e l l u l a r regulation'. A decrease i n plasma osmolality r e s u l t e d i n swelling of flounder erythrocytes ( F u g e l l i , 1967) and Cala (1977) showed that subsequent c e l l volume regulation was accomplished by means of a d i r e c t change i n the permeability of the membrane to Na or K . Net water 102 flow was secondary to net inorganic c a t i o n f l u x . Changes i n the i o n i c composition of plasma must be accompanied by i p n i c s h i f t s within erythrocytes to maintain i o n i c and osmotic equilibrium (Munroe and Poluhowich, 1974; O i k a r i , 1978). There i s also considerable evidence to in d i c a t e that even i n r e l a t i v e l y stable osmoregulators, f l u c t u a t i o n s i n plasma osmolality r e s u l t i n s i g n i f i c a n t shrinking or swelling of red blood c e l l s (Schmidt-Nielsen, 1975). Thus i t appears that successful osmoregulation i n t e l e o s t s undergoing a change i n s a l i n i t y involves both e x t r a c e l l u l a r and i n t r a c e l l u l a r components. In the present study, a drop i n plasma osmolality at the end of the DHA exposure period was accompanied by an increase i n hematocrit. Measurements of the dimensions of red blood c e l l s showed that swelling had indeed occurred. The response of sockeye salmon to sublethal DHA exposure i n fresh water probably represents a complex p h y s i o l o g i c a l r e a c t i o n to the toxicant s t r e s s . Although the actual mechanisms of t o x i c i t y were not investigated, the data suggest that a form of hypoxic stress i s involved which r e s u l t s i n , or i s accompanied by an osmotic i n f l u x of water. Reductions i n plasma osmolality were accompanied by an increase i n s i z e of the red blood c e l l s ; a volume increase which f o r some reason was not regulated. This could r e s u l t from the p a r t i t i o n i n g of DHA in t o the l i p i d components of the red c e l l membrane a l t e r i n g i t s permeability and/or i n t e r f e r i n g with the ion pumping mechanism involved i n the process of volume regulation. A f t e r the toxicant exposure was d i s -continued and the f i s h t r a n s f e r r e d to sea water, the r i s e i n plasma osmolality would f a c i l i t a t e water outflux from the erythrocytes i n t o the hypertonic medium and thus account f o r a lowering of the hematocrit. The depression of hematocrit measured i n exposed f i s h a f t e r 120 h i n sea water may represent a shrinking of red blood c e l l s . As plasma osmolality was i n the normal range at t h i s time,this may be due to inadequate volume reg u l a t i o n a f t e r hyperosmotic 103 stress, perhaps due to an a l t e r a t i o n i n c e l l membrane permeability. Some evidence f o r DHA-induced hypoxia was provided during the course of acute DHA bioassays. Symptoms such as an increased breathing amplitude and frequent coughing indicated that the salmon were undergoing r e s p i r a t o r y d i s t r e s s . This was confirmed during the hypoxia bioassay which demonstrated a dramatic increase i n t o x i c i t y of a normally sublethal dose of DHA brought on by a reduction of dissolved oxygen l e v e l s to 75% saturation. These r e s u l t s suggested that DHA may i n t e r f e r e with the mechanism of oxygen uptake by the blood, leading to hypoxemia. A d d i t i o n a l support f o r t h i s view was provided during the g i l l permeability experiments when i t became apparent that the confinement of f i s h to the tubes increased t h e i r s u s c e p t i b i l i t y to DHA. As dissolved oxygen l e v e l s were maintained i n excess of 90% saturation i n these experiments, i t i s u n l i k e l y that the apparent increase i n t o x i c i t y was due to reduced oxygen a v a i l a b i l i t y . However, i f DHA were to i n t e r f e r e with normal tr a n s f e r of oxygen over the g i l l s , the reduction i n swimming a c t i v i t y of f i s h confined to the tubes would have reduced ram v e n t i l a t i o n and could have l e d to hypoxemia. Because of the added resistance of the f i s h r e t a i n i n g screens, the v e l o c i t y of the water current passing through the tubes was about h a l f of that against which sockeye salmon maintained p o s i t i o n when swimming free i n the donut tanks. Iwama et a l . (1976) observed an increase i n t o x i c i t y of DHA to juvenile coho salmon at reduced a c t i v i t y l e v e l s . In a l l the experiments conducted, sockeye salmon attempted to maintain p o s i t i v e rheotaxis during acute stages of DHA t o x i c i t y . Although one would assume that swimming with the current would have been e n e r g e t i c a l l y much easier, t h i s d i d not occur. A concerted e f f o r t to head i n t o the current may be an adaptive response to increase p a s s i v e l y the volume of water i r r i g a t i n g the g i l l s . 104 Although no information i s a v a i l a b l e on a hypoxia-induced increase of r e s i n a c i d t o x i c i t y , D a v i s (1973) found that sockeye salmon exposed to sublethal l e v e l s of bleached k r a f t m i l l e f f l u e n t (BKME) responded with an increase i n v e n t i l a t i o n volume and coughing rate. In spite of an increase i n oxygen consumption, there was an average 20% reduction i n the oxygen saturation of a r t e r i a l blood. The e f f l u e n t used i n that study had been aerated, neutralized, and f i l t e r e d so that the r e s i d u a l t o x i c i t y was probably a t t r i b u t a b l e to the more p e r s i s t e n t t o x i c components such as r e s i n acids and chlorinated organics. I.H. Rogers (personal communication) l a t e r confirmed the presence of DHA (1-2 mg/L) i n e f f l u e n t from the same pulp m i l l . Davis (1973) suggested that part of the observed reduction i n the ef-f i c i e n c y of gas exchange may have been due to an elaboration of mucus by the g i l l epithelium,as Walden and Howard (1968) reported an increase i n mucus production at the g i l l s of underyearling salmon exposed to neut r a l i z e d BKME. The e f f e c t s of an increase i n d i f f u s i o n distance on f i s h r e s p i r a t i o n have recently been estimated. Based on the assumption that the thickness of the lamellar d i f f u s i o n b a r r i e r was ^ 5 p (Steen, 1971) the elaboration of a 5 um thick layer of mucus on a secondary lamella was cal c u l a t e d to lead to an 81% increase of the d i f f u s i o n resistance to oxygen (Ultsch and Gros, 1979). They also suggested that the presence of t h i s mucus would r e s u l t i n the reduction of the e f f e c t i v e r e s p i r a t o r y water flow by h a l f . The compensatory hyperventilation with i t s attendant oxygen demand at a time when 0^ f l u x i n t o the blood was being r e s t r i c t e d by a d d i t i o n a l mucus corresponds quite c l o s e l y to the observations of increased v e n t i l a t i o n , 0^ consumption and depressed a r t e r i a l P0 2 i n salmon exposed s u b l e t h a l l y to BKME (Davis, 1973). In the present study, no excess mucus production was noted, although an increase 105 in thickness of the order described by TJltsch and Gros (1979) could have occurred in response to DHA exposure. Additional evidence for DHA-induced hypoxia may be suggested from the pattern of plasma electrolyte changes in salmon exposed to DHA in fresh water which followed closely that reported in the literature for hypoxic fis h . At the end of the freshwater exposure period plasma Na was unaltered, Mg was elevated and K + was significantly lowered. An identical pattern was observed by Soivio ejt a l . (1975) during iri v i t ro incubation of rainbow trout blood under f a l l i n g oxygen tension. A progressive rise in hematocrit was accompanied by an influx of K + into the erythrocytes from the plasma. Platner (1950) found that a combination of hypoxic stress and lowered temperature resulted in a marked increase in plasma Mg + + levels in the goldfish which he attributed to a leakage of Mg + + from the erythrocytes into a plasma diluted by water uptake. In the present experiments the lowering of plasma osmolality and elevation of muscle water concentrations indicate a water retention and/or an increase in water uptake from the environment by DHA-exposed fis h . Kidney function As no attempts were made to measure kidney function in the present investigation, the following discussion i s based on an interpretation of the changes observed in plasma electrolytes in the light of the known role of the kidney in hydromineral balance. The kidney plays an indispensable role in osmoregulatory adaptations which determine euryhalinity in teleost fishes such as the salmon. In fresh water, in spite of a relatively low overall permeability, there i s a continuous osmotic influx of water into the fi s h due to the osmotic gradient between the plasma 300 mOsM/kg) and the medium (< 5 mOsM/kg). The kidney 106 counteracts t h i s i n f l u x of water by producing an abundant, strongly hypotonic (to plasma) urine and together with the urinary bladder achieves conservation of f i l t e r e d ions. In sea water the osmotic gradient between the plasma (^  300 mOsM/kg) and the medium 1000 mOsM/kg) i s considerably greater and i n the reverse d i r e c t i o n so that the f i s h tends to become dehydrated. The functions of the kidney and bladder i n sea water are to minimize water l o s s by re-absorption of water and monovalent ions, to produce a urine i s o t o n i c to the plasma, and to excrete d i v a l e n t ions which enter with swallowed sea water (Smith, 1930). The implication that DHA exposure of salmon to fr e s h water r e s u l t s i n hypoxia has already been discussed. One of the adaptive mechanisms by which f i s h respond to hypoxic stress i s that of increasing the fu n c t i o n a l surface area of the g i l l s (Steen and Kruysse, 1964; Holeton and Randall, 1967a). While an increase i n the perfusion of g i l l lamellae f a c i l i t a t e s oxygen uptake, i t concurrently augments the surface area a v a i l a b l e f o r passive osmotic and i o n i c movements. This leads to an increased uptake of water over the g i l l s i n f r esh water (Loretz, 1979). In hypoxic t r o u t Lloyd and Swift (1976) found an increased permeability to water which p e r s i s t e d beyond the time when v e n t i l a t i o n had returned to normal. This increased permeability was r e f l e c t e d i n compensatory increases i n urine flow rate as we l l as a reduction of the active uptake of sodium and chloride by the g i l l s (Swift and Lloyd, 1974). Increased d i u r e s i s combined with a reduction of s a l t reabsorption by the kidney l e d to the excretion of abnormally high concentrations of Na +, K +, Mg + + and C l i n the urine of rainbow tro u t a f t e r hypoxic stress (Hunn, 1969). In the present study, arguments have already been presented to support the view that DHA exposure i n fr e s h water l e d to an increase i n water loading p o s s i b l y v i a the mechanism discussed above. The ra p i d i n f l u x of water across 107 the g i l l s and p o s s i b l y also across the gut may have exceeded the capacity of the kidney to maintain water balance. At high rates of d i u r e s i s , the e f f i c i e n c y of the renal s a l t reabsorptive mechanism i s reduced due to shortened urine residence time both i n the kidney tubules and i n the urinary bladder. As normally between 80-90% of NaCl i s reabsorbed (Lahlou, 1970) t h i s can r e s u l t i n the excretion of urine containing abnormal concentration of e l e c t r o -l y t e s , e s p e c i a l ly c h l o r i d e , such as has been shown to occur a f t e r handling stress and "laboratory d i u r e s i s " (Forster, 1953; Forster and Berglund, 1956; G r a f f l i n , 1931; Holmes, 1961). The c h a r a c t e r i s t i c lowering of plasma C l i n DHA-exposed f i s h during freshwater residence may be due to an unavoidable increase i n the renal excretion of t h i s ion accompanying a compensatory r i s e i n urine production. Another p o s s i b i l i t y , other than a simple overloading of the kidney, which could contribute to a renal i n s u f f i c i e n c y may be a d i r e c t e f f e c t of high l e v e l s of DHA on the kidney function per se. During the sublethal exposure employed i n the present study, DHA l e v e l s i n the kidneys reached 278.1 yg/g. The e f f e c t s of such a concentration are unknown but could p o s s i b l y d i s r u p t normal c e l l u l a r functions. Based on the unit surface area a v a i l a b l e f o r s a l t transport, the c e l l s of the renal tubules are the s i t e s of intense exchanges (Lahlou, 1970), which have been shown to involve Na +-K + ATP-ase (Jampol and Epstein, 1970, Epstein et al.,1969). Enzyme systems involved i n a c t i v e transport may be s e n s i t i v e to high concentrations of DHA. Trump and Jones (1977) described a fundamental c o r r e l a t i o n between the i n h i b i t i o n of a c t i v e transport and u l t r a s t r u c t u r a l changes i n c e l l s of the t e l e o s t nephron. These were r e l a t e d to the a l t e r a t i o n i n permeability of the plasma membrane and mitochondria by a v a r i e t y of t o x i c agents. Studies 108 on organ histopathology were not conducted i n the present study but Fu j i y a (1965) reported kidney tubule necrosis i n marine f i s h taken from waters adjacent to a k r a f t pulp m i l l . DHA-induced kidney dysfunction could have equally important consequences fo r f i s h moving in t o sea water as the successful t r a n s i t i o n of a migrating salmon from a r i v e r to the marine environment depends to a large extent on the a b i l i t y of i t s kidney to assume a very d i f f e r e n t osmoregulatory r o l e , often within a matter of hours. The c r i t i c a l adjustment required of the kidney i n a f i s h faced with increased s a l i n i t y i s a prompt reduction i n urine flow to l i m i t water l o s s . This i s accomplished by a reduction i n glomerular f i l t r a t i o n rate (GFR) (Hickman and Trump, 1969). F a i l u r e to accomplish t h i s quickly w i l l lead to a ra p i d dehydration. A f t e r the t r a n s f e r of DHA-exposed sockeye salmon to sea water, hydration was replaced by a rapid dehydration as demonstrated by a r i s e i n plasma osmolality and a l l measured ions. This was followed by a gradual regulation back down to normal l e v e l s a f t e r 120 h i n sea water. In DHA-exposed f i s h i n f resh water, urine flows could be expected to be elevated to contend with the toxicant-induced hydration. I f such a f i s h i s r a p i d l y t r a n s f e r r e d to sea water, i t i s conceivable that there might be a delay i n the necessary adaptive reduction i n GFR. I n i t i a l l y t h i s would a i d i n the disposal of excess water but i f continued could lead to a rapid dehydration. I t i s probable that the changes observed at the 24 h sampling period do not represent the maximum excursions i n plasma e l e c t r o l y t e s which occurred. Utida (in Fontaine, 1975) observed that during the t r a n s f e r of smolts of the salmon Oncorhynchus masou from FW to SW, maximum plasma 109 osmolality was reached within 8 h and was subsequently regulated back down to normal seawater values. In DHA-exposed sockeye salmon the measurements of plasma e l e c t r o l y t e s i n behaviorally abnormal f i s h revealed that they were not able to regain c o n t r o l of hydromineral balance a f t e r t h i s r a p i d departure from normal e l e c t r o l y t e l e v e l s . The seve r i t y of locomotory d i f f i c u l t y appeared to be r e l a t e d to the extent of t h i s departure from the plasma i o n i c l e v e l s measured i n affected(but s t i l l r e g u l a t i n g ) f i s h . In f i s h which were d r i f t i n g : ( i . e . could not maintain p o s i t i o n against the current) or had j u s t l o s t equilibrium | /plasma e l e c t r o l y t e l e v e l s were grossly increased above both the controls and exposed (but r e s i s t i n g ) groups. Of a l l the ions measured, plasma Mg + + showed the l a r g e s t r e l a t i v e increase and the slowest regulation. This key fi n d i n g provides more d i r e c t support f o r the involvement of the kidney i n DHA-induced hydromineral balance observed i n salmon during seawater adaptation. Beyenbach (1974) found that the tolerance (before l o s s of equilibrium) to Mg + + i n rainbow trout was d i r e c t l y r e l a t e d to the capacity of the ren a l secretory system to maintain magnesium homeostasis. During the adaptation of f i s h to sea water, the temporally second most important function of the kidney becomes that of dival e n t ion secretion; e s p e c i a l l y of magnesium and s u l f a t e which are absorbed across the i n t e s t i n e from ingested sea water. Tubular secretion of Mg + + maintained urine/plasma r a t i o s (U/P) i n the 50:1 to 100:1 range i n seawater-adapted flounder (Hickman, 1968; Lahlou, 1970) coho salmon (Oncorhynchus kis u t c h (Miles, 1971) and rainbow trou t (Beyenbach and Kirschner, 1975). Some DHA-exposed salmon apparently increased water ingestion while s t i l l i n freshwater and thus could have had mucosal p e r m e a b i l i t i e s increased by the d i r e c t contact with the l i p i d - s o l u b l e r e s i n a c i d . Such an increase i n gut permeability could increase the uptake of Mg + + from subsequently 110 swallowed sea water. Although the i n t e s t i n a l absorption route i s generally considered to be the most important, Kirschner et a l . (1974) found that the g i l l epithelium exhibited a low but d e f i n i t e permeability to Mg + +. As e q u i l i b r a t i o n between plasma and sea water according to the electrochemical gradient would r e s u l t i n a 10-20 f o l d increase i n plasma Mg + + concentration (Beyenbach, 1974), i t i s imperative that f i s h maintain a low g i l l permeability to t h i s ion. In view of the changes i n g i l l permeability to water i t may be that p r i o r DHA exposure increased g i l l permeability to Mg + + i n sea water. Even though i t i s not known whether Mg + + uptake v i a the g i l l s increased i n f i s h previously exposed to DHA, the increased entry through the gut alone would probably s u f f i c e to r a p i d l y elevate plasma Mg + + concentrations. The dehydration occurring i n sockeye salmon which were transferred to sea water a f t e r sublethal DHA exposure was shown to be accompanied by a compensatory increase i n sea-water ingestion (Table XII ). Since a net absorption of 44% of the ingested (SW) Mg + + was shown to occur across the i n t e s t i n e of normal trout (Shehadeh and Gordon, 1969) an increase i n sea water ingestion beyond normal l e v e l s could be expected to r a p i d l y r a i s e plasma Mg + + concen-t r a t i o n s . As an increased uptake of Mg + + can only be eliminated by the kidney, and because t h i s ion must be t i g h t l y regulated, the t e l e o s t kidney has developed a powerful tubular Mg + + secretory function. Magnesium i n f u s i o n i n a va r i e t y of euryhaline f i s h species brings about a powerful d i u r e t i c response which p e r s i s t s u n t i l plasma Mg + + l e v e l s return to normal (Bieter, 1933; B r u l l and Cuypers, 1955; Forster, 1953; Hickman, 1968). Natochin et a l . (1970) found s i m i l a r r e s u l t s i n migrating sockeye salmon smolts which they loaded ++ experimentally with Mg . As the rate of urine flow i n sea water i s determined I l l by the i n t e n s i t y of Mg + + excretion, Mg + + loading of salmon can be expected to aggravate dehydration i n sea water (Beyenbach, 1974). Plasma Mg + + l e v e l s are normally regulated within narrow l i m i t s and small v a r i a t i o n s are known to block neuromuscular transmission (del C a s t i l l o and Engbaek, 1954) suggesting that the observed changes i n locomotor performance of DHA exposed f i s h may be r e l a t e d to myoneural blockage by Mg + +. Juvenile pink salmon subjected to the stre s s of scale l o s s i n sea water became un-responsive to v i s u a l and mechanical stimulation a f t e r plasma Mg l e v e l s increased by 63% (personal communication, Dr. L.S. Smith, U n i v e r s i t y of Washington). These symptoms were followed by a progressive p a r a l y s i s . In the present study i d e n t i c a l symptoms were observed i n DHA-exposed., sockeye salmon i n the f i r s t 48 h a f t e r sea water t r a n s f e r . Plasma Mg + + l e v e l s i n these f i s h increased by^50%. F i s h which were d r i f t i n g or moribund had plasma Mg + + l e v e l s 3 to 4 times higher than the exposed (but r e s i s t i n g ) f i s h . Block et a l . (1978) found that plasma Mg + + l e v e l s underwent the greatest increase of a l l e l e c t r o l y t e s measured i n white perch (Morone americana) exposed to chlorine i n estuarine waters. These changes were a t t r i b u t e d to a l t e r a t i o n s i n branchial permeability due to damage to the g i l l epithelium by the toxicant. In the present study, a comparison of the observed changes i n plasma Mg + + l e v e l s with those described i n the l i t e r a t u r e on renal function suggests that DHA exposure of salmon reduces the e f f i c i e n c y of kidney function i n sea water and/or increases g i l l permeability to Mg + + to such an extent that renal excretory mechanisms may become overloaded. Based on the l i t e r a t u r e , ++ elevated plasma Mg l e v e l s may be responsible f or many of the behavioral changes observed i n DHA exposed sockeye salmon during seawater adaptation. 112 Presumably the recovery of f i s h thus affected involves a successful reduction i n GFR and g i l l permeability, thereby reducing water los s and the necessity f o r sea water ingestion. This would be followed by a gradual return of plasma Mg + + l e v e l s to normal. 113 PART I I I . ECOLOGICAL IMPLICATIONS Environmental st r e s s has been defined as "a state produced by any environmental factor which extends the normal adaptive responses of an animal, or which disturbs the normal functioning to such an extent that the chances of s u r v i v a l are s i g n i f i c a n t l y reduced" (Brett, 1958). Pursuing t h i s p h i l o s -ophy further, i n a review of methods used i n the study and i n t e r p r e t a t i o n of sublethal e f f e c t s of toxicants, Sprague (1971) emphasized the need to r e l a t e the often subtle toxicant-induced p h y s i o l o g i c a l changes measured i n the laboratory to successively higher e c o l o g i c a l l e v e l s of i n t e g r a t i o n . In other words, i s the measured change within the normal adaptive range or can i t be expected to contribute to a reduction i n the s u r v i v a l p o t e n t i a l of the e n t i r e organism? This decision poses a s i n g u l a r l y d i f f i c u l t problem as a determi-nation of the e c o l o g i c a l s i g n i f i c a n c e of laboratory findings usually has to be made i n the face of l i m i t e d knowledge of what happens i n the f i e l d and therefore requires a higher than usual l e v e l of personal judgement by the i n v e s t i g a t o r . In the present study, the exposure of j u v e n i l e sockeye salmon to DHA f o r a period of 5 days l e d to s i g n i f i c a n t departures of plasma e l e c t r o l y t e s from "normal" values. Subsequent tr a n s f e r of these f i s h i n t o sea water again resu l t e d i n marked changes i n hydromineral balance i n the d i r e c t i o n of the osmotic and i o n i c gradients. The majority of the f i s h so-affected managed to return plasma e l e c t r o l y t e l e v e l s to c o n t r o l values within 120 h a f t e r the toxicant exposure had been discontinued, i n d i c a t i n g that no permanent reduction i n osmoregulatory capacity had occurred. To suggest whether such a temporary l o s s i n regulatory p r e c i s i o n i s meaningful to the s u r v i v a l of the f i s h one must consider not only the p h y s i o l o g i c a l e f f e c t s but also the e c o l o g i c a l i m p l i c a t i o n s . 114 Special e f f o r t s were made during t h i s study to attempt to c o r r e l a t e changes i n plasma e l e c t r o l y t e l e v e l s , gut water content and muscle water l e v e l s with observed changes i n general behavior of the salmon. Measurement of these parameters at gradually more severe stages of locomotory dysfunction, i . e . d r i f t i n g , equilibrium l o s s , and m o r t a l i t y , confirmed that the more seriously the f i s h were affected, the greater were the departures of the measured va r i a b l e s from the c o n t r o l values. This was e s p e c i a l l y true a f t e r the t r a n s f e r of f i s h to sea water, with plasma Mg + + showing the greatest percent change. Although a cause/effect r e l a t i o n s h i p was not established, based on the l i t e r a t u r e i t i s suggested that the observed locomotory d i f f i c u l t i e s may be r e l a t e d to an interference with neuromuscular function due to high Mg + + concentrations. In addition the high DHA residues occurring i n brain t i s s u e could have l e d to dysfunction at the i n t e g r a t i v e l e v e l and contributed to locomotor d i f f i c u l t i e s through poor muscle co-ordination. One of the f i r s t noticeable e f f e c t s of sublethal DHA t o x i c i t y was a gradual break up of the schooling response. I n i t i a l l y , f i s h - t o - f i s h distances increased and a progressive reduction i n cover response developed. Although schooling i n f i s h i s known to be under v i s u a l c o n t r o l , the l a t e r a l l i n e i s also involved (Pitcher et al.,1976). In the i n i t i a l stages of i n t o x i c a t i o n , salmon which were more widely spaced s t i l l manifested a cover response to v i s u a l stimulation, i n d i c a t i n g that v i s i o n was probably not impaired. At a l a t e r stage, when cover response was completely reduced, a tap on the tank would t r i g g e r an undirected spasmotic swimming behavior suggestive of a h y p e r s e n s i t i v i t y and may i n d i c a t e a dysfunction of the l a t e r a l l i n e system.. Gardner (1975) li n k e d behavioral a l t e r a t i o n s to h i s t o p a t h o l o g i c a l changes i n marine t e l e o s t s exposed to a number of water-borne toxicants. A l o s s i n the a b i l i t y to maintain schooling patterns was r e l a t e d to l e s i o n s i n the o l f a c t o r y 115 epithelium o f A t l a n t i c s i l v e r s i d e (Menidia menidia) exposed to crude o i l i n the laboratory. In the f i e l d , the unnatural behavior and h y p e r s e n s i t i v i t y to mechanical stimulation i n menhaden (Brevoortia tyrannus) c o l l e c t e d i n the e f f l u e n t discharge area of a nuclear generating s t a t i o n were l i n k e d to le s i o n s i n the l a t e r a l l i n e organs. Gardner (1975) also reported that exposure of j u v e n i l e A t l a n t i c salmon (Salmo salar) to pulp m i l l waste both i n laboratory and i n f i e l d studies r e s u l t e d i n l e s i o n s i n o l f a c t o r y organs. The changes i n behavior of DHA-exposed sockeye salmon may have arisen, i n part, from the toxicant-induced changes discussed above. On the other hand, the responses could have ori g i n a t e d i n dysfunction of the nervous system as DHA was shown to accumulate to high l e v e l s i n b r a i n t i s s u e . U n t i l recently the establishment of water q u a l i t y c r i t e r i a has depended almost e x c l u s i v e l y on the r e s u l t s of acute or long-term chronic l i f e c y c l e bioassays (Sprague, 1976). Only a few workers have d i r e c t e d t h e i r a t t e n t i o n to the e c o l o g i c a l s i g n i f i c a n c e of subtle, toxicant-induced behavioral changes. For example, Basch and Truchan (1976) observed g u l l s feeding on small f i s h floundering near the surface during a peri o d of cooling water c h l o r i n a t i o n at a Lake Michigan power plant. While the temporary exposure of a prey species to a p h y s i o l o g i c a l l y sublethal concentration of a chemical toxicant may d i r e c t l y cause l i t t l e more than abnormal behavior, i f greater predation i s the conse-quence then a sublethal e f f e c t has i n d i r e c t l y become l e t h a l to the prey (Goodyear, 1972). These observations form a good example of the concept of " e c o l o g i c a l death". Another example was provided by an experiment reported by F a r r (1977) who exposed grass shrimp (Palaemonetes v u l g a r i s ) s u b l e t h a l l y to Mirex (an organochlorine i n s e c t i c i d e ) and found that a f t e r 13 days there was no d i f f e r e n c e i n s u r v i v a l between co n t r o l s and exposed groups. However, 116 within 24 h of the i n t r o d u c t i o n of predatory p i n f i s h (Lagodon rhomboides) the s u r v i v a l of the previously exposed shrimp dropped p r e c i p i t o u s l y . S i m i l a r r e s u l t s have been demonstrated using other simple f i s h predator/prey systems (Goodyear, 1972; Kania and O'Hara, 1974; S u l l i v a n and Atchison, 1978). Schooling behavior i s w e l l developed i n sockeye salmon smolts and i t may have adaptive value at the estuarine stage of the l i f e c y c l e (Hoar, 1958) or i n the search for food (Eggers, 1976) - Very l i t t l e i s known about the migratory behavior, routes followed,or p h y s i o l o g i c a l and behavioral responses during entry i n t o the sea (Northcote, 1974; Hanamura, 1966). Ricker (1966) suggests that Fraser River sockeye . . . " move out i n t o the offshore p e l a g i c environment rather q u i c k l y " based on t h e i r absence from seine catches. In i n t e r p r e t i n g the s e l e c t i o n behavior of salmon i n a laboratory s a l i n i t y g r a d i -ent, Williams (1969) suggested that sockeye salmon smolts move r a p i d l y out i n t o the S t r a i t of Georgia within several hours of entering the Fraser River Estuary. Williams (1969) also found that smolts from the Cultus Lake run were capable of surviving a d i r e c t t r a n s f e r i n t o sea water. My r e s u l t s support these l a t t e r findings since i n the present experiments c o n t r o l f i s h at the smolt stage maintained t h e i r blood e l e c t r o l y t e concentrations within very o narrow l i m i t s a f t e r t r a n s f e r to 28 /oo sea water. On the other nand, DHA-exposed f i s h could not. I f the changes i n schooling and swimming behavior observed i n the laboratory were to occur i n the f i e l d upon entry i n t o sea water, sockeye smolts could s u f f e r a heavy predation. However,very l i t t l e i s known at the present time about the extent or importance of n a t u r a l predation on normal sockeye salmon smolts on t h e i r seaward migration. Often neglected i n the i n t e r p r e t a t i o n of studies on e l e c t r o l y t e balance of salmonids i s the consideration of s a l i n i t y . Many l a b o r a t o r i e s , the present 117 one included, u t i l i z e coastal sea water ( s a l i n i t y 25-30 ^/oo ) i n t h e i r i n v e s t i g a t i o n s . In the wild, migrating sockeye salmon may move in t o f u l l strength sea water sho r t l y a f t e r leaving the r i v e r and would be exposed to considerably higher s a l i n i t i e s (y 35 °/oo). Boeuf et a l . (1978) found that coho salmon smolts which remained i n an e s s e n t i a l l y homoiosmotic state a f t e r t r a n s f e r i n t o sea water of 30 °/oo s a l i n i t y suffered from a s i g n i f i c a n t hydromineral imbalance when placed i n waters of 35 °/oo s a l i n i t y . Thus DHA-exposed sockeye salmon moving i n t o waters of the S t r a i t of Georgia could be considerably more se r i o u s l y a f f e c t e d than the present study has shown. The laboratory exposure experiments established that salmon can r a p i d l y accumulate DHA i n major organs such as the b r a i n , l i v e r and kidney and that these high residue l e v e l s are probably r e l a t e d to the p h y s i o l o g i c a l dys-function reported i n t h i s t h e s i s . However,DHA depuration rates were not measured, so i t i s not known how long these residues can exert t h e i r t o x i c action, nor i s there any information on the l e v e l s of DHA or other r e s i n acids i n sockeye salmon which have moved down the Fraser River past the pulp m i l l s at Prince George and Quesnel or down the Thompson River past Kamloops. The presence and the p o s s i b l e i n t e r a c t i o n s of DHA with other p e r s i s t e n t toxicants known to be present i n k r a f t m i l l waste have not been studied i n these f i s h nor i n the r i v e r s . The question of a p o t e n t i a t i o n of KME t o x i c i t y by natural f l u c t u a t i o n s i n temperature and dissolved oxygen i n these r i v e r s must be addressed. Results reported i n the present study showed that DHA t o x i c i t y was remarkably increased by a reduction of oxygen to 75% of saturation, a drop well within natural f l u c t u a t i o n s . In view of the demonstrated persistence, bioaccumulation p o t e n t i a l , and known t o x i c i t y of DHA, safe l i m i t s of discharge of t h i s , and p o s s i b l y other r e s i n acids, would better be determined by e s t a b l i s h i n g an " E c o l o g i c a l 118 4/ L i m i t " — f o r these toxicants. This concept takes i n t o account persistence, bioaccumulation, and sublethal thresholds to c a l c u l a t e environmentally safe discharge l e v e l s . Although there appears to be no published information to date on sublethal thresholds of any r e s i n a c i d , sublethal thresholds f o r a v a r i e t y of p h y s i o l o g i c a l and behavioral functions have been determined f o r salmonids exposed to whole k r a f t m i l l e f f l u e n t (Davis, 1976). Perhaps these estimates could be used for e s t a b l i s h i n g E c o l o g i c a l Limits while studies on r e s i n a c i d sublethal thresholds are being conducted. This study has demonstrated that chronic exposure to sublethal DHA concentrations can r e s u l t i n s u b s t a n t i a l toxicant accumulation. Regulatory agencies attempting to set water q u a l i t y c r i t e r i a u t i l i z i n g "safe" concentrations should consider the bioconcentration p o t e n t i a l of r e s i n acids a f t e r t h e i r discharge i n t o r e c e i v i n g waters. When viewed i n the l i g h t of Brett's (1958) d e f i n i t i o n of an environmental st r e s s , sublethal DHA exposure was shown to "disturb the normal functioning" of the sockeye salmon hydromineral homeostatic mechanism and i f the behavior observed i n the laboratory also occurs i n the f i e l d , then i t i s highly probably that the "chances of s u r v i v a l are s i g n i f i c a n t l y reduced". The present study, while l i m i t e d i n i t s scope,underlines the general paucity of information on the p h y s i o l o g i c a l ecology of the sockeye salmon e s p e c i a l l y i n r e l a t i o n to the i n t e r a c t i o n s with pulp m i l l wastes during migration. Furthermore, the study has elucidated sublethal e f f e c t s of DHA on salmonids and p o s s i b l e mechanisms not previously described. While the magnitude and scope f o r deleterious e f f e c t s of such toxicants upon Fraser River stocks has not been d i r e c t l y investigated, the p o t e n t i a l f o r such e f f e c t s upon a s e n s i t i v e l i f e stage of migratory salmonids has been demonstrated. 4/ — E c o l o g i c a l Limit (EL) concept was proposed i n the Netherlands by Canton and Sloof and i s discussed i n van Esch (1978). 119 APPENDIX 1-1. REMOVAL OF DHA FROM FRESH WATER BY SOCKEYE SALMON In t h i s experiment, the acute t o x i c i t y of DHA was investigated i n r e l a t i o n to f i s h loading density during a s t a t i c bioassay. In addition, a n a l y t i c a l methods were developed which permitted the monitoring of the actual concentration of DHA i n the aquarium water. MATERIALS AND METHODS The experiment was conducted using underyearling sockeye salmon which had been hatched at PEI from eggs obtained from the Cultus Lake 1973 brood stock. At the time of the experiment the f i s h were 4 months o l d and had been transferred to laboratory tanks from outdoor f a c i l i t i e s a week e a r l i e r . In the laboratory, they were kept i n tanks provided with a continuous flow of well water at the same temperature as outside (10.5 ±0.5°C). Daily feed-ing with OMP was discontinued 24 h p r i o r to tr a n s f e r of the f i s h to glass aquaria f i l l e d to a volume of 30 L with well water kept at 10.5 ±0.5°C by means of a water bath. An air-stone maintained dissolved oxygen l e v e l s above 90% saturation during the study. A f t e r a 24 h acclimation period, the f i s h were gently t r a n s f e r r e d to i d e n t i c a l t e s t aquaria containing a t h e o r e t i c a l DHA concentration of 1.88 mg/L. One aquarium contained the r e s i n a c i d but no f i s h , to determine the extent of normal adsorption and/or degradation of the toxicant, a second tank was stocked with 5 f i s h (low loading density) and a t h i r d with 10 f i s h (high loading d e n s i t y ) . A fourth aquarium was used as a co n t r o l and contained s i m i l a r d i l u t i o n s of only the DHA c a r r i e r solvents and 10 f i s h . Times to death (as judged by cessation of opercular movement) were recorded, and dead f i s h were weighed and measured. DHA was prepared to a p u r i t y of 94% and a concentrated stock s o l u t i o n was made by d i s s o l v i n g 640 mg of DHA i n 5 mL ethanol followed by 5 mL 5N NaOH 120 and made up to 1000 mL with d i s t i l l e d water. The addition of 94 mL of this solution to the aquarium water yielded a theoretical concentration of 1.88 mg/L DHA. At 24 and 96 h, water samples (500 mL) were taken from both aquaria for determination of DHA concentrations. Water samples were acidified with 1 drop of cone. H2SO4 and extracted with chloroform (4 x 50 mL) i n a l L separatory funnel. The chloroform extracts were evaporated to dryness under vacuum, re-dissolved in diethyl ether, methylated with diazomethane and dried under a stream of nitrogen. Methanol dilutions of these extracts were analyzed by GLC and quantified by comparison of integrated peak areas to a calibration curve based on an external DHA standard. Analyses were done on a Hewlett Packard 7620A gas chromatograph equipped with an integrator and fit t e d with a 1.2 m x 3 mm stainless steel column packed with 10% Silar 5CP on Gas Chrom Q (80-100 M) . Oven temperature was 240°C while injection port and detector (flame ionization) were operated at 250°C. Nitrogen was the carrier gas. RESULTS AND DISCUSSION At the low loading density, a l l fish were dead by 54.3 h (3255 min) whereas at the high loading density only 3 out of 10 fis h died in the f i r s t 39.3 h (2355 min) and no further mortalities occurred during the remainder of the 96 h exposure period (Fig.22). This high rate of survival of fish exposed to 1.88 mg/L DHA can be attributed to their rapid reduction of DHA concentration to a sublethal range (0.86 mg/L) after 24 h followed by a further reduction to 0.29 mg/L after 96 h (Table XVIII).On the other hand, after 24 h at the lower loading density, DHA levels were s t i l l at 1.16 mg/L; a concentration rapidly lethal to the salmon. In addition, a 32% reduction in DHA concentration in the tank containing no fis h indicates that some o High Loading Density 2 .2 g/|_ A Low Loading Density 1.1 g/L The e f f e c t s of f i s h loading density in. a s t a t i c bioassay on the acute t o x i c i t y of DHA (1.88 mg/L) to j u v e n i l e sockeye salmon. Table XVIII. The e f f e c t of f i s h loading density on concentration of DHA i n aquarium water Loading density DHA concentration g/L mg/L 24 h 96 h 0 1.44 0.98 1.1 1.16 0.77 2.2 0.86 0.29 " i n i t i a l t h e o r e t i c a l concentration 1.88 mg/L. 123 adsorption or degradation took place. These r e s u l t s showed that a reduction i n DHA l e v e l s i n the water occurred and that t h i s reduction was greatly enhanced by the presence of f i s h . However because DHA may not have been a c t u a l l y i n s o l u t i o n or may have been removed by simple adsorption to the tank walls or to the surface of the f i s h , experiments were conducted to answer these questions. These experiments are described i n the r e s t of Appendix I. 124 APPENDIX 1-2. EFFECTS OF FILTRATION ON DHA RECOVERY FROM FRESH WATER Although DHA recovery experiments confirmed that the r e s i n a c i d was present i n the water at close to the t h e o r e t i c a l concentrations,they d i d not in d i c a t e whether the toxicant was i n so l u t i o n . The r e s i n a c i d could have been adsorbed to p a r t i c u l a t e s present i n the water or could simply have been present i n suspension and as such might have been bound or not f r e e l y a v a i l -able to the f i s h . As these p a r t i c u l a t e s should be r e a d i l y f i l t e r a b l e , a comparison of DHA recovery from f i l t e r e d and u n f i l t e r e d solutions was made. METHODS Replicate solutions of DHA were made up using laboratory well water as used i n bioassay experiments. A concentrated stock s o l u t i o n (238.5 mg/L DHA) which had been prepared f o r a bioassay was the source of the r e s i n a c i d and the same d i l u t i o n r a t i o as used i n continuous-flow bioassays was u t i l i z e d (3 mL stock/500 mL water). Four i d e n t i c a l solutions were prepared i n 1000 mL glass b o t t l e s which were stoppered, shaken to ensure mixing and then l e f t f o r o 36 h i n a water bath at 10.5 ±0.5 C. Two of the samples were then a c i d i f i e d with 1 drop cone. H2SO4 and extracted with chloroform as previously described while the other two were suction f i l t e r e d through a 0.22 ym Nucleopore f i l t e r before e x t r a c t i o n . The extracts were then analyzed by GLC as before. RESULTS AND DISCUSSION F i l t r a t i o n had no e f f e c t on the recovery of DHA from the water (Table XIX). As the o r i g i n a l t h e o r e t i c a l concentration was 1.35 mg/L DHA, and as the mean percentage recovery was 86.9% some loss d i d occur during the experiment, probably by adsorption to the glass i n the b o t t l e s as w e l l as some l o s s during the extraction process. As "apparent s o l u b i l i t y " has been o p e r a t i o n a l l y defined by f i l t r a t i o n through a 0.45 ym membrane f i l t e r (Suffet and Faust, Table XIX. Comparison of concentrations of DHA recovered from f i l t e r e d and u n f i l t e r e d water samples. Replicate DHA concen t r a t i o n samples m 9 / L F i l t e r e d A U n f i l t e r e d A 1.17 Recovery % 86.7 1.18 87.4-1.18 87.4 1.16 85.9 ' O r i g i n a l concentration 1.3 5 mg/L. 1972) and i n the present experiment a 0.22 um f i l t e r was used, i t was concluded that the DHA was indeed dissolved and as such should be f r e e l y a v a i l a b l e to the f i s h . 127 APPENDIX 1-3. ESTIMATE OF THE DIRECT SOLUBILITY OF DHA IN FRESH WATER Although DHA may be considered as being water insol u b l e according to the chemical d e f i n i t i o n (Grant, 1944), the extent of t h i s s o l u b i l i t y may nevertheless be s u f f i c i e n t to be of t o x i c o l o g i c a l and therefore b i o l o g i c a l s i g n i f i c a n c e . To t e s t t h i s hypothesis, the d i r e c t aqueous s o l u b i l i t y of DHA was determined by gas chromatographic methods. METHODS Dehydroabietic a c i d (95% purity) c r y s t a l s were ground to a fi n e powder using a mortar and p e s t l e and three samples were weighed on an electrobalance (Perkin Elmer Autobalance Model AD^2) to 0.01 mg and then'transferred to 500 mL well water (pH 6.76) i n 1000 mL glass b o t t l e s . Magnetic s t i r bars were used to keep the mixture i n suspension. A f t e r 24 h, the contents of each b o t t l e , now at room temperature (20°C) were f i l t e r e d through a 0.45 um f i l t e r . A f t e r a c i d i f i c a t i o n , the f i l t r a t e s were extracted and analyzed as previously described. RESULTS AND DISCUSSION The recovery of DHA from the water showed that the r e s i n a c i d was d i r e c t l y soluble i n w e l l water to an average of 3.3 mg/L (Table XX ). Nyren and Back (1958) determined the t o t a l s o l u b i l i t y (sum of ionized and unionized DHA) to be 6.6 mg/L while f o r the unionized a c i d i t was 4.9 mg/L and demonstrated that the s o l u b i l i t y was a function of pH. These authors suggested that the aromatic r i n g of DHA rendered i t more h y d r o p h i l i c than other r e s i n acids such as a b i e t i c . The r e s u l t s of the present experiment suggest that DHA may be capable under c e r t a i n conditions, of d i s s o l v i n g d i r e c t l y i n water to a l e v e l acutely t o x i c to salmonids. While chemically " i n s o l u b l e " , dehydroabietic a c i d can be considered t o x i c o l o g i c a l l y "soluble" 128 Table XX . Concentrations of DHA d i r e c t l y soluble i n f r e s h water at pH 6.76 and 20°C. Replicate Concentration of DHA Concentration of DHA samples i n o r i g i n a l mixture i n s o l u t i o n mg/L mg/L 29.78 3.15 B 31.68 3.20 33.26 3.61 as the free a c i d . As discussed i n the Introduction, the p r e c i s e chemical status of DHA i n r e c e i v i n g waters has not been established although i t i s usually discharged as a sodium s a l t . The exact chemical species w i l l then be determined by r e c e i v i n g water chemistry. In a l l bioassay experiments conducted during the present study, DHA was prepared as the sodium s a l t before addition to the aquarium water. 130 APPENDIX 1-4. UPTAKE OF DHA FROM FRESH WATER AND ITS DISTRIBUTION IN THE TISSUES OF JUVENILE SOCKEYE SALMON AND A MATURE RAINBOW TROUT INTRODUCTION The f i s h - l o a d i n g density experiment described i n Appendix 1-1 showed that DHA was removed from the water by the presence of the salmon, however t h i s removal could be accomplished by simple adsorption to the surface of the f i s h or by an actual absorption i n t o the f i s h . I t i s known that i n f i s h e s the entry of a toxicant across the g i l l membranes i s re l a t e d to i t s p a r t i t i o n c o e f f i c i e n t and i t s pKa (Hunn and A l l e n , 1974). Dehydroabietic a c i d i s a weak organic a c i d of very low aqueous s o l u b i l i t y but which i s f r e e l y soluble i n l i p i d solvents such as chloroform. Although no published information on the p a r t i t i o n c o e f f i c i e n t of DHA could be found i n the l i t e r a t u r e , the p a r t i t i o n c o e f f i c i e n t s of a wide spectrum of compounds are i n v e r s e l y r e l a t e d to t h e i r aqueous s o l u b i l i t y and i n turn determine biomagnification and l i p o p h i l i c storage (Chiou e_t a l . , 1977; Neely e t a l . , 1974; Yang and Sun, 1977). Based on i t s s o l u b i l i t y p roperties and at the pH of w e l l water DHA could be expected to pass r e a d i l y through the g i l l epithelium (McLeay e_t a l . , 1979) . A f t e r entering the vascular system, l i p i d soluble compounds are thought to dissolve i n plasma l i p o p r o t e i n s (Fromm, 1970) and are then d i s t r i -buted throughout the body by the blood (Holden, 1962) where they may be deposited i n t i s s u e s of high l i p i d content (Branson et a l . , 1975; Freed et a l . , 1976). As a preliminary experiment (unpublished) showed that DHA was indeed taken up and concentrated from the water by sockeye salmon during an acute bioassay, two d e t a i l e d experiments were performed to measure the extent of DHA accumulation during sublethal exposure and to determine the t i s s u e 131 d i s t r i b u t i o n of these residues. MATERIALS AND METHODS F i s h and Test Conditions Two separate experiments were conducted, one with sockeye salmon and one with a sing l e rainbow trou t (Salmo g a i r d n e r i ) . Sockeye salmon smolts were obtained from the Great Central Lake (Vancouver Island) migration and kept i n the PEI outdoor holding f a c i l i t y f o r 2 months p r i o r to use. A sing l e mature female rainbow trou t (fork length 47 cm, 1520 g wet weight) was used f o r the trout experiment and was part of a stock obtained from a commercial supplier i n Mission, B.C. These f i s h were 2+ years o l d and had been held at PEI f o r an a d d i t i o n a l 18 months before the experiment. The f i s h were held at 11.5 ±0.5°C i n well water (see Table I ) . In the salmon experiment, 10 f i s h (15-20 g) were transferred to the laboratory donut tanks r e c e i v i n g well water at the same temperature and were held there f o r a further 72 h before the toxicant exposure began. The f i s h were not fed during the acclimation period or during the 120 h exposure period. Ten salmon were exposed to 0.65 mg/L DHA i n one tank and another 10 served as controls i n a second tank; a t h i r d tank contained the si n g l e r a i n -bow trout. A continuous current of 15 cm/sec was maintained and the flow of wel l water at a rate of 500 mL/min provided a 90% replacement time of ^  4 h (Sprague, 1969). The f i s h loading density f o r the salmon was 3.6 L water/g fish/day and 0.47 L/g/day f o r the tr o u t . Toxicant The DHA toxicant was prepared and metered i n t o the incoming water system by procedures described prev i o u s l y . A GLC check of the DHA concentra-t i o n a c t u a l l y present i n the water showed that ^ 90% of the t h e o r e t i c a l dosage had been attained. The pH of the bioassay water a f t e r addition of the 132 stock s o l u t i o n was 6.96 f o r the DHA and 6.97 f o r the c o n t r o l tank. The water temperature was maintained at 11 ±0.5°C and d i s s o l v e d oxygen > 90% saturation. F i s h t i s s u e preparation At the conclusion of the exposure period, the salmon were anesthetized with 200 mg/L MS-222, while the t r o u t was stunned by a blow on the head. Blood was c o l l e c t e d from severed caudal peduncles of both species. Blood from each salmon was spotted on a microscope s l i d e f o r the preparation of blood smears and a f t e r c e n t r i f u g a t i o n the hematocrit was measured and the plasma was pooled f o r the determination of osmolality. In the salmon the packed red blood c e l l s were pooled f o r the determination of DHA residues while f o r the rainbow trout, DHA residues were determined i n a sample of whole blood. The g a l l bladder b i l e was c o l l e c t e d from both species using 3 mL B-D Vacutainers. The l i v e r , kidney, spleen, g i l l , gut and brain were r a p i d l y removed from both species and i n the salmon, the remainder was termed the "carcass". Individual t i s s u e s were pooled and freeze-dried to constant weight. For the trout, a d d i t i o n a l samples of ova and l a t e r a l muscle were taken but the carcass was not analyzed. In the salmon, the e n t i r e branchial basket was u t i l i z e d while i n the t r o u t only the g i l l filaments were used. The t r o u t muscle sample was removed from the d o r s o - l a t e r a l muscle mass immediately p o s t e r i o r to the dorsal f i n and the skin was removed before weighing. The large mass of the various t i s s u e s i n the t r o u t permitted accurate wet weights to be determined before freeze-drying so that the % water could be determined. In salmon, where the wet weights of pooled t i s s u e s were not measured, wet weights were approximated by using the salmon dry t i s s u e weights which had been measured and applying the % water obtained f o r the t r o u t data. In the case of salmon b i l e , erythrocytes, g i l l s and carcass, the r e l a t i o n s h i p 133 between dry and wet weight was established from co n t r o l samples. Chemical Extraction Dry t i s s u e samples were ground by mortar and p e s t l e with sodium s u l f a t e , a c i d i f i e d with a drop of 10% H^SO^, packed on a chromatographic column (1 cm x 20 cm or 2 cm x 30 cm, depending on sample volume) and extracted with 100-200 mL p e s t i c i d e grade methylene chloride (Burdick & Jackson Laboratories Inc., Michigan, USA). The extracts were evaporated to dryness under vacuum, redissolved i n d i e t h y l ether, methylated with diazomethane, and sui t a b l e hexane d i l u t i o n s were then analyzed by GLC. Analysis of two control l i v e r samples spiked with DHA yi e l d e d 92.2 and 99.3% recovery f o r t h i s e xtraction procedure. B i l e i n the Vacutainer v i a l s was decanted 5 times with methylene chlo r i d e , a c i d i f i e d , evaporated to dryness, methylated and then analyzed by GLC. A n a l y t i c a l Procedure The presence of DHA was confirmed i n hexane d i l u t i o n s of the f i s h e xtracts by gas chromatography-mass spectrometry (GC-MS), using a Hewlett Packard HP 5992A f i t t e d with a 1.8 m x 6 mm glass column packed with 3% OV-101 on Supelcoport 80-100 mesh. The oven temperature was programmed from 150 to 250°C at 8 C/min a f t e r an i n i t i a l isothermal hold of 2 min. o Detector and i n j e c t i o n port temperature was 250 C and helium was the c a r r i e r gas. An external DHA standard was used f o r comparison of the mass spectra. Ions were scanned from 40 to 400 mass u n i t s with a sing l e ion being monitored at m/e 239 CEnzell and Wahlberg, 1969). Quantitative determination of DHA was made by GLC on a Hewlett Packard HP 5700 f i t t e d with a flame i o n i z a t i o n detector u t i l i z i n g a s p l i t r a t i o of 100:1 and a 30 m x 0.2 mm column wall-coated with OV-101. The oven temperature was programmed from 124 to 205 UC at 8 C/min a f t e r an i n i t i a l isothermal hold of 2 min. Detector and i n j e c t i o n port temperature was o 250 C and helium was the c a r r i e r gas. DHA was used as an external standard. DHA Residue C a l c u l a t i o n Resin acid residues are expressed both as ug DHA/g dry t i s s u e and wet t i s s u e i n order to f a c i l i t a t e the comparison of concentration f a c t o r s (tissue : water). The salmon wet t i s s u e weights are estimates derived from measured dry weights as previously described. The whole body residues f o r the sockeye salmon were determined by summing the dry weight of a l l the tissues to re c o n s t i t u t e the " o r i g i n a l " f i s h . As the % water for the carcass was known from c o n t r o l samples, the o r i g i n a l t o t a l wet weight of the exposed f i s h could be back-calculated from the dry weights. The t o t a l DHA residue recovered from the various t i s s u e s was summed and divided by the wet weight of the f i s h . For the trout, the t o t a l body residues were ca l c u l a t e d by summing the i n d i v i d u a l t i s s u e DHA residues and d i v i d i n g by the o r i g i n a l t o t a l f i s h wet weight. Estimates were made f o r blood and muscle according to proportions i n the l i t e r a t u r e . T o t a l blood residues were estimated on the basis of a blood volume of 3% of body weight (Smith, 1966) while t o t a l "muscle" residues were ca l c u l a t e d on the basis of a muscle mass of 67% of body weight (Stevens, 1968). As the trout was gravid and heavily laden with eggs, "body weight", f o r the purposes of these c a l c u l a t i o n s , was taken as the t o t a l wet weight minus eggs. RESULTS Both f i s h species accumulated DHA from the water. On a whole body, wet weight basis, the sockeye salmon DHA residues were 19.2 mg/kg while the rainbow trou t contained 22 mg/kg. The DHA d i s t r i b u t i o n i n various t i s s u e s 135 i s shown f o r salmon (Fig.23) and f o r rainbow trout (Fig.24) expressed on a wet weight and dry weight basis ( l e f t and r i g h t ordinate scale, r e s p e c t i v e l y ) . A l l c a l c u l a t i o n s which follow are based on wet t i s s u e weights. As shown i n F i g . -23, the highest o v e r a l l concentration of DHA was found i n the salmon b i l e (647.3 yg/g); t h i s y i e l d s a bile/water r a t i o of 996. Of the salmon organs, the b r a i n contained the l a r g e s t amount (619.8 yg/g) follow-ed by the kidney (278.1 yg/g) and l i v e r (262.5 yg/g) y i e l d i n g bioconcentration r a t i o s of 954, 428, and 404 r e s p e c t i v e l y . The carcass, which consists of the o r i g i n a l body minus the t i s s u e s l i s t e d to the r i g h t i n Fig.23 and there-fore includes the head, skeleton, muscle, skin etc., contained 7.7 yg/g DHA. On a wet weight basis, the i n t e r n a l organs and t i s s u e s represent only 17% of the t o t a l f i s h weight yet contained 29% of the t o t a l body residue. The l i v e r and kidney accounted f o r 40% of the organ DHA burden. The rainbow trout residue t i s s u e d i s t r i b u t i o n (Fig.24 ) shows the l i v e r (290.6 yg/g) kidney (.182.5 yg/g) and brain (154.3 yg/g) contained the highest residues, y i e l d i n g bioconcentration f a c t o r s of 447, 281 and 238 r e s p e c t i v e l y . The b i l e contained 17.9 yg/g r e s u l t i n g i n a bile/water r a t i o of 27.5. For the trout, the l i v e r and kidney accounted f o r 56% of the t o t a l organ DHA burden. While the whole body bioconcentration f a c t o r s f o r sockeye smolts and rainbow trou t amounted to 30 and 34 r e s p e c t i v e l y , i t can be seen that i n i n d i v i d u a l organs, f o r example i n the l i v e r , DHA was present at 400 times the 0.65 yg/mL l e v e l present i n the water during the 120 h exposure period. B i l e samples of both the salmon and trout were found to contain at l e a s t three metabolic d e r i v a t i v e s of the parent DHA molecule. One of the d e r i v a t i v e s was i d e n t i f i e d as methyl abietatetraenoate. DHA was not detected i n any of the t i s s u e s taken from c o n t r o l f i s h . 136 r4.2 o x -5! z7-g t— z -LU 8 -m A. s -UJ 1 1 CO cr, < < O DRY | | W E T cr > UJ UJ a co UJ I r— cr LU 3 CJ < cr CO 4.0 3.8 3.6 co O 3.4 x N: 3.2 =<• :1.4 z o H.2S r-Z LL) 1.0 IOB" CO r0.6^  LU m 0.4' -0.2 < X Q Figure 23. D i s t r i b u t i o n of dehydroabietic a c i d i n pooled t i s s u e s of sockeye salmon smolts a f t e r a 5 day laboratory exposure to 0.65 mg/L DHA. . . 137 o X CO 2 O DRY WET -1.2 1.0 p X < rx LU o o LU " CO CO : 2^ LU < D CO LU LU LU 2 o D O O _ i CD 3 < LU Z I LU LU n Q. co 5 % 3 >-LiJ > < rx -0.6 I o o LU Z3 CO -0.4 £ >• rx Q < I Q 0.2 Figure 24; Tissue d i s t r i b u t i o n of dehydroabietic acid i n a rainbow trou t a f t e r a 5 day laboratory exposure to 0.65 mg/L DHA. 138 DISCUSSION Dehydroabietic a c i d i s a weak organic a c i d which,based on i t s aqueous s o l u b i l i t y (Appendix 1-3), can be c l a s s i f i e d chemically as water "insoluble" (Grant, 1944). On physicochemical grounds DHA at the pH of well water would be expected to p a r t i t i o n r a p i d l y from the water, across g i l l e p i t h e l i a , i n t o the blood and from there be d i s t r i b u t e d to the various l i p i d pools with-i n the body (Hunn and A l l e n , 1974; McLeay et a l . , 1979). This i s substan-t i a t e d by the presence of high DHA residues i n l i p i d extracts of t i s s u e s i n salmon and trout i n the present study. No published information i s a v a i l a b l e on l e v e l s of DHA i n i n d i v i d u a l organs of f i s h . Hagman (1936) i n analyses of moribund f i s h c o l l e c t e d down-r i v e r from a s u l f a t e pulp m i l l , found a p o s i t i v e (colorimetrie) reaction f o r r e s i n acids i n extracts of l i v e r , pancreas, kidney and mucus, with an e s p e c i a l l y strong reaction f o r the " l i q u i d surrounding the brain". The present study has shown that the b r a i n of the sockeye salmon accumulated the highest DHA residues of any organ. Tomiyama (in F u j i y a 1965) suggested that penetration of r e s i n acids i n t o f i s h t i s s u e s may have l e d to chronic t o x i c e f f e c t s such as the h i s t o p a t h o l o g i c a l changes reported by F u j i y a . F u j i y a noted a v a r i e t y of n e c r o t i c changes i n the kidney, i n t e s t i n e , pancreas and g i l l s of f i s h taken from waters r e c e i v i n g k r a f t pulp m i l l waste, with p a r t i c u l a r l y serious e f f e c t s on the l i v e r (Fujiya 1961, 1965). In the present work, the l i v e r contained among the highest DHA residues i n the two salmonid species tested. The high l e v e l of DHA i n the b i l e of salmon as well as the presence of more polar metabolites i n both f i s h species suggests a h e p a t o b i l i a r y excretion route f o r t h i s r e s i n a c i d . In f i s h e s , t h i s route of xenobiotic 139 excretion i s now well established, with the l i v e r p l a y i n g a fundamental r o l e i n biotransformation of l i p i d soluble compounds in t o more polar, water soluble forms which are then excreted v i a the b i l e (Adamson and Sieber 1974; Klaassen 19751. A number of p e s t i c i d e s , polychlorinated biphenyls, phenols, and detergents have been i s o l a t e d from the b i l e of a v a r i e t y of f i s h species (Lech et a l . , 1973; Gakstatter, 1968; Melancon and Lech, 1976; Statham et a l . , 1973;Lech, 1973; T o v e l l et a l . , 1975). Smith (1971) reported that among the requirements f o r extensive b i l i a r y excretion, a balance between p o l a r i t y and non-polarity, molecular weight of 300-400, and the presence of an e a s i l y i o n i z a b l e group are important physicochemical c h a r a c t e r i s t i c s . DHA, with a molecular weight of 300 and a carboxyl group would appear to f i t these requirements. Hydroxylation by the l i v e r would increase molecular weight, make the r e s i n a c i d more polar and thus f a c i l i t a t e i t s b i l i a r y excretion. B i l e flow i s also an important determinant of the rate at which many compounds are cleared from the plasma and excreted i n the b i l e (Tuttle and Schottelius, 1969). During the exposure to DHA, the sockeye salmon developed a marked yellowish tinge which was p a r t i c u l a r l y noticeable i n the membranes of the p e c t o r a l and p e l v i c f i n s as well as i n the plasma. These symptoms were found to be due to a form of jaundice caused by a DHA-induced r i s e i n plasma b i l i r u b i n . These r e s u l t s are discussed i n depth i n Appendix I I I . Jaundice i n mammals i s known to r e s u l t when hepatic clearance of b i l i r u b i n i s reduced by acute l i v e r disease or obstruction of the b i l e duct (Nosslin,1960). B i l e i s known to be t o x i c to human l i v e r parenchyma when released i n t o the t i s s u e s during acute obstructive jaundice (Sherlock, 19681; s i m i l a r l y Hendricks et a l . , (1976) reported that rainbow trou t b i l e was highly c a u s t i c to t r o u t l i v e r t i s s u e . In the present experiment, the 140 f a i l u r e or overloading of the h e p a t o b i l i a r y system i n salmon exposed to DHA may have contributed to the accumulation of DHA residues to t o x i c l e v e l s i n various organs. Presumably when uptake exceeds metabolism and excretion, accumulation of DHA and/or b i l e i n the l i v e r may occur and r e s u l t i n l o c a l i z e d t i s s u e necrosis. The rainbow trout d i d not e x h i b i t jaundice. I t s much larger s i z e may have enabled i t to store or metabolize more of the DHA before i t s t o x i c e f f e c t s become apparent; the r e s u l t s of preliminary acute bioassays (unpub-lished) indicated that larger f i s h may be more r e s i s t a n t to the acute t o x i c e f f e c t s of DHA. The sublethal exposure of f i s h to a toxicant can lead to i t s accumulation at high concentrations i n various i n t e r n a l organs which subsequently undergo his t o p a t h o l o g i c a l changes. This mode of t o x i c action has been well documented fo r p e s t i c i d e s and r e l a t e d chemicals accumulated by f i s h i n the laboratory (Buhler et a l . , 1969; E l l e r , 1971; Kruzynski, 1972; Mathur, 1962; Mount, 1962) or i n the w i l d (Johnson, 1968; Kennedy et al^., 1970) . In many cases r a p i d and pronounced changes occur i n the l i v e r (Couch, 1975) although frequently most of the organ systems are a f f e c t e d (see Walsh and R i b e l i n , 1975 f o r review). The accumulation of heavy metals such as mercury and cadmium has been shown to r e s u l t i n damage to f i s h kidney tubules (Trump et a l . , 1975) as well as i n renal l e s i o n s and l i v e r and gonad degeneration ( T a f a n e l l i and Summerfelt, 1975). These authors concluded that the h i s t o p a t h o l o g i c a l changes and subsequent p h y s i o l o g i c a l dysfunction were caused d i r e c t l y by the accumulation of large organ residues a f t e r sub-lethal exposure. 141 Although h i s t o p a t h o l o g i c a l observations were not attempted i n the present study, i t would be i l l u m i n a t i n g to determine whether the high DHA residues can be linked to i n t e r n a l organ histopathology; such as reported by F u j i y a (1961) i n f i s h exposed to k r a f t m i l l waste i n the v i c i n i t y of a coastal pulp m i l l . Renal damage may be one of the main contributing factors to the hydromineral imbalance observed i n the DHA-exposed f i s h . The calculated whole body wet weight DHA residues obtained i n t h i s study compare favorably with those I found i n a preliminary (unpublished) study. Using a semi-quantitative technique (Mahood and Rogers, 1975) to achieve the separation of i n t e r f e r i n g f a t t y acids from DHA p r i o r to GLC analysis I measured whole body residues of 27.3 mg/g DHA i n sockeye salmon which had died during freshwater exposure to 1.1 mg/L DHA. This represents a b i o -concentration of ^  25 x that t h e o r e t i c a l l y present i n the water. Fox et a l . (1977) exposed 200 g rainbow trout to d i l u t i o n s of k r a f t m i l l waste (KME) and found whole f i s h residues of DHA approximately 20 times the DHA concentrations present i n the d i l u t e d e f f l u e n t . Based on t h e i r f i g u r e s , a 48 h continuous exposure of trout to KME containing a mean concentration of 0.67 ug/mL DHA re s u l t e d i n residues of 10 yg/g. In the present study, an exposure of 120 h to.0.65 yg/mL DHA resu l t e d i n residues of 22 yg/g f o r the rainbow t r o u t and 19.2 yg/g for the sockeye salmon. I f the uptake rate of DHA f o r rainbow trout as reported by Fox - et a l . (1977). was l i n e a r , then a f t e r 120 h t h e i r f i s h should have contained approximately 25 yg/g DHA. This estimate compares favorably with the r e s u l t s obtained i n the present experiment. I f such a comparison i s v a l i d , then the time f o r e q u i l i b r a t i o n of the uptake rate would exceed 5 days. Technically, the synthesis of r a d i o - l a b e l l e d DHA would g r e a t l y s i m p l i f y the estimation of uptake and depuration rates of t h i s r e s i n a c i d . 142 APPENDIX 1-5. THE ACCUMULATION OF DHA IN SALMON VIA THE DIETARY ROUTE-EVIDENCE OF ACCUMULATION IN THE SALMON FOOD ORGANISM Anisogammarus confervicolus. INTRODUCTION In addition to absorbing DHA d i r e c t l y from the water, salmon may feed on organisms which have accumulated the r e s i n a c i d above ambient l e v e l s . The uptake and bioconcentration of some p e s t i c i d e s by members of the various trophic l e v e l s of the aquatic food chain has been well documented (Ha t f i e l d , 1969; Johnson et_ a i . , 1971; Schoenthal, 1963) and i n some cases, t h i s route i s thought to be the major source of contamination f o r f i s h i n natural waters (Macek and Korn, 1970). F i e l d studies on feeding habits have established the general importance of estuarine amphipods i n the d i e t of juvenile salmon (Goodman and Vroom, 1972; Levings, 1973). Healey (1978) found that amphipods dominated the d i e t of sockeye salmon smolts sampled i n Georgia S t r a i t , B.C. and the amphipod Anisogammarus confervicolus was found to be the most important food item i n the d i e t of juvenile chinook salmon O. tshawytscha within the area of influence of a coastal pulp m i l l (Kask and Parker, 1972). As an increase i n the abundance of these food organisms has been observed i n the v i c i n i t y of pulp m i l l o u t f a l l s and log storage areas ( B i r t w e l l , 1978; Harger and Nassichuk, 1974; Waldichuk and Bousfield, 1962) i t i s conceivable that juvenile salmonids may be "attracted" i n t o waters containing r e l a t i v e l y high concentrations of t o x i c waste components. In addition, i f food organisms such as amphipods were to accumulate these toxicants above ambient l e v e l s , there would a r i s e the p o t e n t i a l f o r b i o -concentration of the more p e r s i s t e n t t o x i c components by f i s h u t i l i z i n g t h i s contaminated food source. 143 As DHA i s known to be one of the more p e r s i s t e n t toxicants present i n k r a f t pulp m i l l waste, a laboratory study was conducted to determine whether t h i s r e s i n a c i d was taken up from the water by a representative salmon food organism; the amphipod A. confervicolus. MATERIALS AND METHODS Amphipods were c o l l e c t e d from troughs draining the outdoor f i s h holding tanks at PEI.. Water i n the troughs ( s a l i n i t y ^ 10 °/oo ) supported a t h i c k growth of mussels (Mytilus edulis) among which l i v e d the amphipods and the isopod Gnorimosphaeroma oregonensis. No e f f o r t was made to separate the amphipods from the mussel mass and approximately equal amounts of the mixture were placed i n t o two glass c y l i n d e r s 9.2 x 40.7 cm (2.76 L) with the ends covered by f i b e r g l a s s mosquito net t i n g . Each c y l i n d e r was then l a i d f l a t on the bottom of separate donut tanks i n which the r e c i r c u l a t i n g pump maintained a continuous water current of ^ 15 cm/sec. Approximately 30 g of the alga Fucus vesiculosus c o l l e c t e d from the beach was added to each cy l i n d e r to provide a d d i t i o n a l s h e l t e r . A f t e r a 48 h acclimation period, the amphipods were exposed to 0.4 mg/L DHA i n brackish water (10 ±1 °/oo ; 10.5 ±0.5°C; pH 6.97; disso l v e d oxygen>90% saturation) under continuous-flow conditions. A flow rate of 740 mL/min provided a 95% replacement time of 4.5 h (Sprague, 1969). The con t r o l tank received the d i l u e n t solvents without the r e s i n a c i d . At the conclusion of the 120 h exposure period, the c y l i n d e r s were l i f t e d out of the tanks, the contents emptied into a tray and the amphipods were picked out with forceps. During the s o r t i n g procedure, the amphipods were placed i n t o a tray containing uncontaminated brackish water. A f t e r s o r t i n g was complete, they were placed on a screen, r i n s e d with deionized water, b l o t t e d and then frozen r a p i d l y with l i q u i d CC^. Each amphipod mass 144 was weighed and then freeze d r i e d to determine the r e l a t i o n s h i p between wet and dry weight. A f t e r grinding with Na2S0^, the sample was extracted with methylene chloride and processed i n the same manner as previously described f o r the f i s h samples. A f t e r GLC a n a l y s i s , the r e s u l t s were expressed on both a wet weight and dry weight basis. RESULTS Shortly a f t e r t h e i r t r a n s f e r into the laboratory donut tanks, the amphipods appeared to have s e t t l e d i n t o the various crevices and the mussels were a c t i v e l y feeding. During the course of the experiment, no dead or behaviorally abnormal amphipods were observed and the accumulation of f e c a l p e l l e t s downstream of the glass c y l i n d e r s indicated that active feeding continued throughout the exposure period. The r e s u l t s show that the amphipods contained DHA at a l e v e l 21 x that t h e o r e t i c a l l y present i n the water (Table, XXI). On a whole body wet-weight basis, t h i s f i g u r e compares favorably with the 29.5-fold concentration of DHA by sockeye salmon exposed to 0.65 mg/L of the r e s i n a c i d f o r the same length of time. As both exposures were of r e l a t i v e l y short duration the data do not provide information on the shape of the uptake curve so that DHA residues at equilibrium are not known. No DHA was detected i n the c o n t r o l samples. DISCUSSION This experiment i n d i c a t e s that there e x i s t s a p o t e n t i a l f o r bio-accumu-l a t i o n of DHA v i a the d i e t a r y route, however the estimate of i t s r e l a t i v e importance must await uptake and a s s i m i l a t i o n studies. As f i s h - f o o d organisms such as amphipods may be exposed to DHA from t h e i r own food supply 145 Table XXI. DHA residues i n the amphipod Anisogammarus confervicolus exposed to 0.4 mg/L DHA for 120 h i n sea water of s a l i n i t y 10 °/oo . T o t a l Weight of sample Wet g Dry g N DHA Wet yg/g Dry yg/g Concentration ' F a c t o r 2 Exposed 6.8400 1.9760 133 8.39 29.03 21 Control 10.7136 3.0793 208 ND 1 Estimate based on i n d i v i d u a l wet weight (51.6 mg) c a l c u l a t e d from the pooled wet weight of 100 amphipods. 2 Wet weight basis r e l a t i v e to maximum t h e o r e t i c a l concentration i n the water. 146 as well as d i r e c t l y from the water, such studies could best be done i n a model ecosystem u t i l i z i n g several members of the aquatic food chain. Nevertheless, recent studies have shown that the regions around pulp m i l l s may be a t t r a c t i v e to j u v e n i l e salmonids because of the presence of a more r e a d i l y a v a i l a b l e food supply. Under laboratory conditions, the addition of d i l u t e KME to a r t i f i c i a l freshwater stream communities l e d to an enhancement of the biomass of the amphipod Crangonyx ( E l l i s , 1967) and i t appears that s i m i l a r e f f e c t s do occur i n the f i e l d . Kelso (1977) suggested that the intense aggregation of the f i s h community near a k r a f t pulp m i l l o u t f a l l i n Lake Superior was. due to the extreme abundance of benthic invertebrates adjacent to the p o i n t of entry of the e f f l u e n t plume. He indicated that the feeding response to a high benthic biomass may over-r i d e any avoidance r e a c t i o n to the e f f l u e n t . Increased numbers of juvenile chinook salmon i n the area adjacent to a c o a s t a l pulp m i l l at Port A l b e r n i B.C. may have been due to such an enhancement of the food supply ( B i r t w e l l , 1978). F i s h thus "attracted" i n t o regions of increased food supply would be exposed to elevated l e v e l s of toxicants i n the water as well as i n the d i e t . Although the consequences of consuming contaminated f i s h food organisms i n the wild are unknown, Tokar (1968) conducted a laboratory growth study on juvenile chinook salmon fed on t u b i f i c i d worms (oligochaetes). Salmon fed on worms which had previously been exposed to f u l l strength KME for 24 h consumed equal amounts of food but had much lower growth rates than the controls. This was a t t r i b u t e d to a s t r i k i n g reduction i n the e f f i c i e n c y of food u t i l i z a t i o n . As the f i s h were kept i n e f f l u e n t - f r e e water these e f f e c t s strongly suggest a sublethal t o x i c action by some component accumulated from the e f f l u e n t and absorbed by the f i s h . 147 Since species such as chinook, coho and chum salmon can spend extended periods of time feeding i n estuarine habitats which are also being used for the discharge of pulp m i l l wastes ( B i r t w e l l , 1978; Davis et a l . , 1978; S i b e r t and Kask, 1978), the ingestion of DHA-contaminated food organisms could be of considerable s i g n i f i c a n c e to these as well as to other salmonid species. I f the rate of t r a n s i t of sockeye salmon smolts through other estuaries i s as rapid as that thought to occur i n the Fraser River Estuary, (Williams, 1969), i t i s u n l i k e l y that the consumption of DHA-contaminated amphipods would play a s i g n i f i c a n t p art i n the buildup of DHA residues. On the other hand, while s t i l l i n the r i v e r , sockeye smolts feed p r i m a r i l y on aquatic insects (Goodman, 1964) so that t h i s food source could be a p o t e n t i a l source of DHA contamination. I t i s not known whether the p o t e n t i a l f o r bio-accumulation of DHA suggested by t h i s preliminary study i s r e a l i z e d i n the f i e l d s i t u a t i o n . However circumstantial evidence indicates that t h i s may indeed be the case. Brownlee et a l . (1977) found DHA i n bottom sediments at a distance of up to 15 km from the o u t f a l l of a k r a f t pulp and paper m i l l i n Lake Superior. Such sediment-bound DHA may be taken up by benthic organisms and subsequently translocated to bottom-feeding f i s h . Brownlee and Strachan (1977) found DHA i n the sucker (Catastomus); a bottom feeder, and i n yellow perch (Perca  flavescens) which feed on aquatic invertebrates as well as small f i s h e s . I t i s p o s s i b l e that these f i s h accumulated DHA d i r e c t l y from the water i n the region of the pulp m i l l , but based on the amphipod data obtained i n the present study, there appears to be l i t t l e reason to exclude the food chain route from a c o n t r i b u t i n g r o l e i n the buildup of DHA i n wild f i s h . 14! APPENDIX I I . BACTERIAL KIDNEY DISEASE(BKD) Salmonid b a c t e r i a l kidney disease(BKD) i s caused by a genus of Corynbacterium and a f f e c t s a l l f i v e species of P a c i f i c salmon, the A t l a n t i c salmon, as well as most of the commonly cultured t r o u t species. I t may be present i n the chronic stage and progress slowly with no apparent symptoms for much of the year but frequently reaches e p i z o o t i c proportions i n the early spring months. Because i t i s b a s i c a l l y asymptomatic i n the chronic stages, i t usually passes unnoticed u n t i l some of the more severely i n f e c t e d f i s h begin developing external symptoms or s t a r t dying. As BKD i n f e c t i o n s are highly r e s i s t a n t to a n t i m i c r o b i a l drugs (Suzomoto et a l . , 1977) no treatment has yet been found, so that when the disease arises i n aquaculture operations the e n t i r e i n f e c t e d stock i s r o u t i n e l y destroyed. As a r e s u l t , BKD i n f e c t i o n s have s e r i o u s l y a f f e c t e d production i n a number of U.S. t r o u t hatcheries (Snieszko et a l . , 1955) and are c u r r e n t l y one of the most serious problems f a c i n g P a c i f i c salmon cu l t u r e both i n hatcheries and marine net pens. Although most prevalent i n f i s h c ulture operations, i t i s l i k e l y that BKD also a f f e c t s natural stocks of salmon although the consequences on marine su r v i v a l are unknown. The disease has been found i n "wild" rainbow t r o u t i n B.C. (Evelyn et a l . , 1973) and r e c e n t l y i n a "wild" chinook salmon i n Puget Sound, Washington ( E l l i s et a l . , 1978). In 1977, 11 of 41 coho salmon sampled returning to the Capilano River (West Vancouver, B.C.) as spawning adults were found to be i n f e c t e d with BKD (G.E. Hoskins, P a c i f i c B i o l o g i c a l Station, personal communication). Recent evidence suggests that resistance to BKD i n coho salmon may be g e n e t i c a l l y determined (Suzomoto e_t a l . , 1977). 149 Once the disease i s established, i t s course and s e v e r i t y appear to be aff e c t e d by a v a r i e t y of f a c t o r s such as water temperature and hardness, season, degree of crowding and d i e t . In a consideration of water chemistry i n 37 U.S. salmonid hatcheries, Warren (1963) found that as the constituent load of the water decreased, the s e v e r i t y of corynbacterial kidney disease increased. However an e t i o l o g i c r e l a t i o n s h i p was not established. Wedemeyer et a l . (1976) suggested that increased m o r t a l i t i e s i n s o f t waters may be due to increased energy requirements f o r osmoregulation. Although BKD i s termed kidney disease, i t i s a c t u a l l y a systemic i n f e c t i o n and can a f f e c t most of the v i t a l organs, although the hematopoietic t i s s u e of the kidney and spleen are among the f i r s t t i s s u e s i n f e c t e d (Wood and Yasutake, 1956; Snieszko et a l . , 1955). In more acute stages, the i n -f e c t i o n leads to necrosis of the e n t i r e kidney and can extend to the g i l l s , l i v e r , spleen, eyes, musculature and a n t e r i o r g a s t r o i n t e s t i n a l t r a c t ( B e l l , 1961) . Young and Chapman (.1978) described u l t r a - s t r u c t u r a l changes i n the glomerulus and renal tubules of BKD i n f e c t e d brook t r o u t (Salvelinus  f o n t i n a l i s ) which were interpreted as signs of i r r e v e r s i b l e c e l l i n j u r y . The progressive destruction of kidney t i s s u e can be expected to lead to osmoregulatory dysfunction and i t has been postulated that eventual death may be due to renal i n s u f f i c i e n c y (Bendele and Klontz, 1975). Wood and Yasutake (1956) suggested that the complex and extensive morphological changes observed i n other organs were probably not a d i r e c t r e s u l t of impaired renal function as the excretory system was one of the l a t t e r t i s s u e s to be affected by the disease, but that each organ was a f f e c t e d d i r e c t l y by the b a c t e r i a . These authors a t t r i b u t e d the often-reported edema to damage to the c i r c u l a t o r y system, e s p e c i a l l y the mesenteric blood vessels. A reduction i n hematocrit, 15C hemoglobin, and plasma p r o t e i n i n i n f e c t e d f i s h was i n t e r p r e t e d as a symptom of osmoregulatory dysfunction (Hunn, 1964; Suzumoto et a l . , 1977). While i t i s d i f f i c u l t to determine how f a r along the disease has progressed before the excretory function of the kidney i s s e r i o u s l y a f f e c t e d i t i s known that i t s hematopoietic function i s reduced e a r l y i n the i n f e c t i o n . The progression from chronic s u b c l i n i c a l to the manifested _stages i s therefore accompanied by a lower-ing of hematocrit (Suzomoto et a l . , 1977; Iwama, 1977). The Occurrence of BKD During E l e c t r o l y t e Balance Experiments In the present study, i n d i c a t i o n s of the presence of a chronic BKD i n f e c t i o n i n the sockeye salmon smolts became apparent during blood sampling i n Expt. 1. Several f i s h had lowered hematocrits and one showed a swollen abdomen which i s one of the v i s i b l e symptoms of i n f e c t i o n . However no m o r t a l i t i e s were observed i n the stock holding tanks containing ^ 2000 smolts i n the week p r i o r to or during Expt. 1. Hematocrits obtained i n Expt. 2, which followed immediately, i n d i c a t e d that the disease was progressing more r a p i d l y and a chronic m o r t a l i t y (.1-2 f i s h per day) began i n the stock holding tanks at t h i s time. Shortly a f t e r , a confirmation of the presence of BKD i n the salmon was obtained through the Diagnostic Service of the F i s h Health Program, P a c i f i c B i o l o g i c a l Station, Nanaimo, B.C. and the e n t i r e stock was destroyed. At t h i s time tank m o r t a l i t i e s remained low (2-3 f i s h per day i n a stock o f ^ 2 0 0 0 ) , t y p i c a l of the chronic stage of BKD i n f e c t i o n . As i t was impossible to judge the s e v e r i t y of the chronic BKD i n f e c t i o n i n f i s h used i n Expts. 1 and 2 u n t i l a f t e r the blood sampling was completed, f i s h showing lowered hematocrit were judged to be i n an advanced stage of chronic i n f e c t i o n and were deleted from the s t a t i s t i c a l consideration of the data. This procedure was based on two assumptions: A) that as the disease pro-gressed i n severity, hematocrit dropped and B) that the incidence of i n f e c t i o n 151 was normally d i s t r i b u t e d i n the f i s h used i n the experiments. Support f o r the f i r s t assumption was provided above, while the b a s i s f o r the second assumption was ensured by a random sampling of f i s h from the stock tanks f o r use i n Expts. 1 and 2. Hoffman (1963), using the properties of the normal d i s t r i b u t i o n , de-scribed a g r a p h i c a l method which separates c l i n i c a l l y healthy from diseased persons. By using arithmetic p r o b a b i l i t y paper, a normal d i s t r i b u t i o n can be transformed i n t o a s t r a i g h t l i n e by p l o t t i n g cumulative frequencies, expressed as percentages of the t o t a l frequency against the end points of the clas s i n t e r v a l s . When treated i n t h i s manner, a composite d i s t r i b u t i o n comprising c l i n i -c a l l y "normal" and " s i c k " components can be g r a p h i c a l l y s p l i t i n t o two by eye f i t t i n g a s t r a i g h t l i n e to the component which represents the c l i n i c a l l y normal data with maximum weight being given to the points around 50%. Normal l i m i t s are then a r b i t r a r i l y defined to be the values which g r a p h i c a l l y enclose 95% of those obtained by t e s t i n g c l i n i c a l l y "normal" subjects. In the present experiment therefore, f i s h were defined as "diseased" i f t h e i r hematocrits f e l l i n the lower 2.5% of the c l i n i c a l l y "normal" values. Separate curves were p l o t t e d using the hematocrits of a l l the "con t r o l " f i s h f o r Expt. 1 and Expt. 2, the lower l i m i t s were e s t a b l i s h e d and then applied to eliminate f i s h from both c o n t r o l and experimental groups having hemato-c r i t s below the lower l i m i t s . In Experiment 1 the lower l i m i t was 26.8% while f o r Experiment 2 i t was 25.2%. The data f o r the f i s h which were thus eliminated are presented i n Table XXII and XXIII r e s p e c t i v e l y . Due to the progressive nature of BKD, t h i s screening method was consider-ed to eliminate f i s h which were i n the advanced chronic or severe stages of a disease which may compromise osmoregulatory performance. This screening Table XXII.Size, hematocrit and plasma i o n i c composition of f i s h deleted from data of Expt. 1 because of a suspected advanced i n f e c t i o n of b a c t e r i a l kidney disease. Sampling c Group Fi s h s i z e Hematocrit Osmolality Chloride Sodium Potassium Calcium Magnesii Time Fork length Wet weight (hours) cm g % mOsm/kg mEg/1 mEq/1 mEq/1 mEq/1 mEq/1 24 C 14.5 33.2 26.4 298 126.0 159.7 2.78 5.57 1.82 48 E 15.8 36.4 26.3 298 140.0 152.0 3.23 4.90 3.79 72 E 14.8 31.7 25.9 264 113.0 150.5 3.21 6.31 1.52 96 C 14.2 28.1 20.8 284 129.5 148.8 3.33 5.27 1.59 120 E 14.6 29.2 25.5 298 135.5 161.0 3.41 5.25 1.68 Based on hematocrit < 26.8% ^Sockeye salmon smolts were exposed to sea water a f t e r a 120 h exposure to 0.65 mg/L DHA i n fresh water. "C=control, E=exposed. to Table XXIII. Size, hematocrit and plasma i o n i c composition of f i s h deleted from data of Expt.2 because of a ' suspected advanced i n f e c t i o n of b a c t e r i a l kidney disease. Sampling Group Fish s i z e Hematocrit Muscle Gut Osmolality Chloride Sodium Potassium Calcium Magnesium Time Fork length Wet weight Water Water (hours) cm g % % % mOsm/kg mEq/1 mEq/1 mEq/1 mEq/1 mEq/1 0 C 16.4 47. 7 21. 7 75. 69 77. 93 290 113 . 0 148. 2 4. 47 5. 48 1.56 C 17.2 52. 9 17. 7 75. 38 79. 72 274 114 . 0 144. 2 3. 66 6. 13 1.56 24 C 16.5 45. 5 21. 3 71. 81 73. 61 300 130. 5 148. 1 3. 29 5. 53 2.65 C 16.4 40. 5 23. 73. 31 76. 77 334 131, .0 170.9 4. 36 7. 30 4.11 c 13.6 28. 1 39. o d 71. 29 72. 76 350 162. 5 172. 6 4. 17 3. 61 2.09 48 c 14.0 28.5 20. ,2 73. 55 77. 27 294 129. ,5 156. 7 4. ,74 5. ,49 1.59 c 15.7 41. ,2 22. .7 75. ,25 79. 07 304 131. ,5 • 158. ,3 3. ,45 5. ,10 1.91 C 13.8 28. 6 . 19. ,9 74. 47 81. 17 306 127. ,5 160. ,0 4. ,45 5. .51 1.73 72 C 16.4 48. ,7 20. .6 74. ,95 77. ,98 306 129 .0 150. .1 4. .26 5. .13 1.58 C 14.9 33. .4 19. .8 74. .50 79. .44 304 122, .5 161. .0 3, .99 5. .91 1.89 C 14.7 29. ,1 22. .8 75. .92 79. ,46 292 129 .0 154. ,3 3, .97 5, .58 1.95 E 17.7 58. ,0 24. .0 74. .90 84. .17 302 138, .5 157. .6 4. .03 5, .03 1.76 E 16.6 43. .1 23. .2 75. .03 80. .03 232 100 .0 122, .4 2 .59 4, .18 2.45 96 C 17.6 53, .0 9. .3 74, .67 77, .15 304 132 .0 153, .7 3 .25 5 .13 1.76 C 15.8 41. .1 18, .6 74, .93 80, .24 296 133 .5 158, .8 4 .02 5 .26 1.51 E 16.0 36, .0 21 .4 75 .12 79 .71 304 134 .0 144 .3 5 .53 4 .85 2.20 E 14.9 37, .3 20 .6 74 .75 81, .39 282 131 .5 150 .2 3 .62 5 .13 2.43 120 c 14.1 27 .7 24 .5 75 .15 77 .15 316 130 .0 156 .9 4 .47 6 .74 1.75 C 16.0 37 .8 18 .4 75 .88 79 .43 300 124 .5 146 .2 4 .25 5 .95 1.72 C 15.9 40 .8 22 .5 74 .50 80 .31 298 129 .5 153 .4 4 .55 5 .29 1.61 E 15.0 37 .3 21 .9 81 .20 75 .41 282 127 .5 150 .7 4 .06 4 .43 1.62 Based on hematocrit < 25.2%. Sockeye salmon smolts were exposed to sea water a f t e r a 120 h exposure to 0.65 mg/L DHA i n fresh water. C=control, E=exposed. ^Eliminated on basis of BKD symptoms; f l u i d f i l l e d abdomen. 154 method could not eliminate c h r o n i c a l l y i n f e c t e d f i s h so that the r e s u l t s of Expt. 2 may be viewed as an i n t e r a c t i o n of disease and the toxicant. The Interaction of BKD with the T o x i c i t y of DHA The presence of a chronic BKD i n f e c t i o n appeared to increase the suscep-t i b i l i t y of sockeye salmon to DHA t o x i c i t y (Fig.25). In Expt. 3 conducted with healthy f i s h , only 2 out of the 72 exposed f i s h died; one near the end of the freshwater toxicant exposure period and the second a f t e r 65.5 h i n the seawater recovery phase. In Expt. 1, i n which the BKD i n f e c t i o n was f i r s t detected, the disease appeared to contribute to an increase i n the l a t e n t t o x i c i t y of DHA, that i s , m o r t a l i t i e s began only a f t e r the f i s h had been i n sea water f o r 24 h with a t o t a l of 6/60 exposed f i s h dying i n t h i s period. The "mortality" curve shown f o r Expt. 2 was generated i n a s l i g h t l y d i f f e r e n t manner than f o r Expts. 1 and 3 i n which a c t u a l times to death were recorded. In Expt. 2 four f i s h were c o l l e c t e d a f t e r death but eight f i s h were sampled i n a "moribund" condition to permit the determination of % water * i n muscle and gut while s t i l l a l i v e . Based on past observations, these f i s h would have died within 3-4 hours but for the purposes of the construction of the t o x i c i t y curve, sampling time was taken to be time to death. The a d d i t i o n of several hours would have made a small d i f f e r e n c e i n the l o c a t i o n of the l i n e ; s h i f t i n g i t s l i g h t l y to the r i g h t (Fig. 25) . Three f i s h were sampled at the "inverted" stage ( f i s h which had l o s t equilibrium) but were not used to p l o t the t o x i c i t y curve. In Expt. 2, i n which the BKD i n f e c t i o n was reaching an advanced chronic stage i n some f i s h , the combined stress loading due to the disease and toxicant was shown by m o r t a l i t i e s occurr-ing both e a r l i e r and i n greater numbers, as i l l u s t r a t e d i n F i g . 25. That the slope of the mortality curve does not break u n t i l 24 h i n t o the seawater recovery period indicates a persistence i n the enhancement 155 Figure 25. The extent of salmon mo r t a l i t y during the three e l e c t r o l y t e balance experiments (Expts. 1, 2 and 3) i n which sockeye salmon smolts were exposed to sea water a f t e r a 120 h exposure to 0.65 mg/L DHA i n f r e s h water. 156 of the sublethal t o x i c i t y of DHA. A f t e r the break, two more f i s h died f o r a t o t a l of 15/75. Evidence that the BKD i n f e c t i o n per se or i n combination with the s a l i n i t y s t r e s s was not l e t h a l i s provided by the t o t a l absence of cont r o l m o r t a l i t y i n Expt. 1 and only 1 mo r t a l i t y out of 72 control f i s h i n Expt. 2 i n which the disease was more advanced. In the present context "acute t o x i c i t y " i s defined as that occurring during the 96-h toxicant exposure period. The "sublethal" exposure used f o r the e l e c t r o l y t e balance experiments was purposely chosen to cause n e g l i g i b l e mortality i n an a d d i t i o n a l 24-h exposure to DHA. The mo r t a l i t y data confirm that t h i s design was achieved and that depending on the severity of the BKD i n f e c t i o n , t h i s "sublethal" exposure s h i f t e d c l o s e r to the "acute" l e v e l of exposure and the i n t e r a c t i o n of stresses of disease/toxicant/sea water contributed to an increase i n the l a t e n t t o x i c i t y of DHA. This would amount to a s h i f t to the l e f t of the acute t o x i c i t y curve (Fig.9) which was the basis f o r the choice of a concentration of DHA which would be "safe" f o r 120 h. 157 APPENDIX I I I . PLASMA BILIRUBIN INTRODUCTION During the course of exposure to DHA, ju v e n i l e sockeye salmon were seen to develop a jaundiced appearance characterized by a yellowish tinge which became p a r t i c u l a r l y noticeable i n the membranes of the paired f i n s . As jaundice i n mammals i s brought about by a buildup of b i l i r u b i n i n the blood, plasma b i l i r u b i n l e v e l s of exposed f i s h were compared to those of controls. In vertebrates, b i l i r u b i n i s a natural end-product of the catabolism of hemoglobin, formed i n the r e t i c u l o e n d o t h e l i a l system, delivered to the blood stream and cleared by uptake i n the l i v e r c e l l s . Two types of b i l i r u b i n are recognized: unconjugated (UCB) and conjugated (CB) b i l i r u b i n . B i l i r u b i n normally c i r c u l a t i n g i n the blood stream (UCB) i s p r o t e i n bound and i n -soluble i n water. In the l i v e r , i t undergoes a conjugation and i s thus rendered soluble before excretion i n t o the b i l e . This d i f f e r e n t i a l s o l u b i l i t y forms the basis of the " d i r e c t " - soluble (CB) vs. " i n d i r e c t " - insoluble(UCB) t e s t f o r b i l i r u b i n and the r e l a t i v e proportions of CB/UCB are used to characterize the type of jaundice involved. For example, a r i s e i n serum UCB without a concomitant r i s e i n CB can in d i c a t e an increase i n blood destruction or hemolysis but that the he p a t o b i l i a r y system i s s t i l l excreting b i l i r u b i n i n a normal fashion. As normal serum contains n e g l i g i b l e to very low amounts of CB, i t s r i s e i s i nterpreted as a sign of a he p a t o b i l i a r y disorder such as acute parenchymal disease or b i l i a r y obstruction (Gray, 1961; Nosslin, 1960; Wintrobe, 1961). To determine whether the apparent jaundice was r e l a t e d to elevated b i l i r u b i n l e v e l s , both CB and UCB concentrations were measured i n sockeye salmon smolts i n two experiments (Expt. PB-1 and PB-2). 158 MATERIALS AND METHODS In both experiments a 5-day exposure of smolts to 0.65 mg/L DHA i n fresh water was followed by terminal blood sampling and a determination of plasma b i l i r u b i n l e v e l s . In Expt. PB-1 b i l i r u b i n was determined i n blood pooled from 10 f i s h which had been exposed during the DHA t i s s u e residue experiment (Appendix 1-4). When elevated plasma b i l i r u b i n l e v e l s were confirmed, the exposure was repeated using 20 f i s h . In addition to the controls f or each experiment, b i l i r u b i n was also measured i n a sample of plasma pooled from 5 salmon taken from the laboratory stock tank during preliminary work. The sockeye salmon smolts used i n these experiments were from the same stock as used i n the DHA t i s s u e residue experiment and holding, acclimation, toxicant exposure and blood sampling protocol was the same as described i n the General Methods. Af t e r the measurement of hematocrit (Hct) of each f i s h , the blood plasma was pooled f o r measurement of osmolality (mOsM), t o t a l (CB+UCB) and d i r e c t (CB) b i l i r u b i n as well as plasma i r o n (Fe). Plasma Fe was measured to determine whether the b i l i r u b i n e m i a could be a t t r i b u t e d to increased hemo-globin breakdown as occurs i n hemolytic jaundice (Smith, 1973). Plasma b i l i r u b i n was measured by the modified diazo procedure developed by Michaelsson (1961) f o r the determination of serum b i l i r u b i n i n newborn i n f a n t s . However, rather than using 1 mL of 1/10 d i l u t e d serum, _ 50 uL of undiluted plasma was added to the reagents halved i n volume. Absorbance was measured at 600 nM on a Pye Unicam or a G i l f o r d 2400 Spectrophotometer. To check the accuracy of the method, a c a l i b r a t i o n curve was constructed using t o t a l b i l i r u b i n l e v e l s given i n human serum c a l i b r a -t i o n references (General Diagnostics, C a l i b r a t e I, I I , I I I ) . As t h i s showed 159 that the absorption law was being followed, the c a l i b r a t i o n constant of 43 was used as described by Michaelsson (1961). Plasma i r o n was measured i n pooled samples of blood taken from salmon i n Expts. PB-1 and 2, as well as from a sin g l e rainbow trou t (1400 g) during the development of the a n a l y t i c a l procedures. Preparative methods followed those described f o r human serum (Perkin Elmer, 1971) and plasma i r o n was measured by Atomic Absorption Spectrophotometry using an air-acetylene flame at 248.3 nm UV. RESULTS No m o r t a l i t i e s were observed i n e i t h e r experiment during the 5-day exposure period. While blood plasma of con t r o l f i s h was e s s e n t i a l l y c l e a r , that of the DHA-exposed f i s h developed the c h a r a c t e r i s t i c yellow tinge. The r e s u l t s of the blood chemistry analyses are shown i n Table XXIV f o r Expt. PB-1 and i n Table XXV for Expt. PB-2. Both experiments show s i m i l a r r e s u l t s . Plasma b i l i r u b i n l e v e l s are elevated i n f i s h exposed to DHA, with the d i r e c t (CB) accounting f o r 81.6% and 69.7% of the t o t a l b i l i r u b i n (Expts. PB-1 and 2 r e s p e c t i v e l y ) . There appears to be very l i t t l e change i n plasma i r o n , while hematocrit was elevated and osmolality lowered by the r e s i n a c i d exposure. In the blood plasma pooled from the 5 salmon taken from the stock holding tank, conjugated b i l i r u b i n was not detectable while t o t a l b i l i r u b i n was 0.30 mg%. The plasma of the single rainbow trout contained 30 ug% i r o n . DISCUSSION An increase i n plasma b i l i r u b i n can be caused by increased blood destruc t i o n or by the reduction i n the capacity of the l i v e r to remove the pigment from the blood and excrete i t i n the b i l e . However, the l i v e r i s generally Table XXIV. Blood chemistry of sockeye salmon smolts exposed to 0.65 mg/L DHA f o r 5 days i n Expt. PB-1. Pooled Plasma Blood B i l i r u b i n Iron Osmolality Hematocrit T o t a l D i r e c t mg% yg% mOsm/kg % Exposed N=10 2.06 1.68 46 282 49.0 ±1.55 Controls N=10 0.19 0.11 42 290 41.7 ±0.69 Size range 15-20 g bMean ±SE S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p< 0.05 ( t - t e s t ) Table XXV. Blood chemistry and s i z e of sockeye salmon smolts exposed to 0.65 mg/L DHA for 5 days i n Expt.PB 2. Pooled Plasma B i l i r u b i n Iron Osmolality T o t a l D i r e c t mg% Blood Hematocrit yg% mOsm/kg Fish Fork Wet length weight x ±SE cm g Exposed N=20 2.28 1.59 38 288 52.4 ±0.8 13.7 ±0.3 22.41 ±1.57 Controls N=18 0.47 ND 38 297 43.0 ±0.6 14.0 ±0.2 22.0 ±1.01 Not detectable ^Differs s i g n i f i c a n t l y from co n t r o l p<0.05 (t-t e s t ) 162 considered to have a large reserve capacity and an increase i n plasma b i l i r u b i n can usu a l l y be lin k e d to a dysfunction i n the hepatic excretion of the pigment (Wintrobe, 1961). Nevertheless, the l e v e l s of plasma b i l i r u b i n have been selected as a measure of increased Hb degradation i n rainbow trout and coho salmon exposed to formalin (Wedemeyer,1971). Hemoglobin i s broken down into globin, i r o n and b i l i r u b i n . In the rainbow trout, most of the i r o n i s bound to the plasma p r o t e i n t r a n s f e r r i n (Fromm, 1977), however during increased red blood c e l l destruction, t h i s binding capacity may be exceeded and lead to increased plasma Fe l e v e l s . The method used i n the present study measured the l e v e l s of unbound plasma Fe and the r e s u l t s i n d i c a t e no measurable e f f e c t of DHA on t h i s parameter i n sockeye salmon. The range of plasma i r o n values (38-46 yg%) i s lower than the mean of 55 yg% obtained f o r the rainbow trout (Fromm, 1977). In the present study i t i s u n l i k e l y that the observed increase i n plasma b i l i r u b i n was due to increased red blood c e l l destruction or hemolysis. The exposure of salmon to DHA i n fresh water i n v a r i a b l y l e d to an elevation, not a lowering of hematocrit, no change or a s l i g h t drop i n plasma K + and v i r t u a l l y no change i n plasma Fe l e v e l s ; both of which could be expected to r i s e as a r e s u l t of increased erythrocyte destruction. The observed r i s e i n plasma b i l i r u b i n can therefore be a t t r i b u t e d to a reduction i n the e f f i c i e n c y of i t s excretion. The r e l i a b l e measurement of trace amounts of b i l i r u b i n i n plasma i s d i f f i c u l t and considerable work has been done towards the development of methodology, mostly because of the importance of accurate determinations i n in f a n t s s u f f e r i n g from neonatal jaundice. The method of Michaelsson (1961) i s considered to be the most r e l i a b l e (With, 1968) and was applied to the 163 measurement of salmon b i l i r u b i n i n t h i s study. Due to the use of d i f f e r e n t methods, widely varying l e v e l s of plasma b i l i r u b i n have been reported f o r f i s h . Sakai and Kawazu (1978) reported 0.025 mg% f o r the carp (Cyprinus . carpio) and 0.032 mg% f o r the rainbow trout. S a t i a et a l (1974) and Buckley (1976) reported 0.05 mg% f o r rainbow trou t and coho salmon r e s p e c t i v e l y while Wedemeyer (1971) found plasma b i l i r u b i n l e v e l s of 0.6 mg% f o r rainbow trout and 1.6 mg% f o r coho salmon. In the present study, t o t a l plasma b i l i -rubin l e v e l s i n c o n t r o l sockeye salmon averaged 0.33 mg%. The sample of plasma pooled from 5 salmon taken from the stock tank y i e l d e d 0.30 mg%. The accompanying very low or non-detectable l e v e l s of conjugated b i l i r u b i n i n co n t r o l samples are consistent with the mammalian l i t e r a t u r e (Gray, 1961; Wintrobe, 1961). When there i s increased blood destruction, the conjugated form of b i l i r u b i n constitutes l e s s than 15% of the t o t a l ; higher proportions are usually a t t r i b u t e d to the r e g u r g i t a t i o n of the conjugated b i l i r u b i n glucuronide by the l i v e r (Michaelsson, 1961). In salmon exposed to DHA, there was a dramatic r i s e i n the t o t a l b i l i r u b i n , of which the bulk (70-82%) was a t t r i b u t e d to the conjugated form. This constitutes further evidence against increased blood destruction and these symptoms therefore can be interpreted as a r e s u l t of obstructive jaundice. Two main types of obstructive jaundice are recognized: A) Mechanical -due to extra-hepatic obstruction of b i l e flow and B) Parenchymatous - due to intra-hepatic obstruction stemming from the d i s t o r t i o n of l i v e r c e l l a r c h i t e c t u r e by l o c a l necrosis of l i v e r c e l l s (Gray, 1961). Extra-hepatic obstruction leads to the forced retention of b i l e i n the l i v e r , distending the b i l i a r y passages and eventually rupturing the b i l e c a p i l l a r i e s . This r e s u l t s i n the passage of b i l e i n t o the sinusoids and back i n t o the blood. As the 164 b i l e i s t o x i c to human l i v e r parenchymal c e l l s , i t s leakage out of the b i l e c a n a l i c u l i can lead to l o c a l necrosis (Sherlock, 1968). Hendricks et a l . (1976) reported that rainbow trout b i l e was highly c a u s t i c to trout l i v e r t i s s u e . The jaundice observed i n salmon s u b l e t h a l l y exposed to DHA could be of both types. In f i s h which swallowed large amounts of fresh water the increased hydrostatic pressure of a t u r g i d stomach may have reduced b i l e flow. In f i s h i n which no excess water intake was observed, intra-hepatic obstruction i s suggested and may be r e l a t e d to l i v e r histopathology. Residue analyses showed that the l i v e r had among the. highest residues of DHA of a l l the ti s s u e s investigated (Fig.23 and Fig.24). The high concentrations of DHA i n the l i v e r , the jaundice and the suggested importance of the hepato - b i l i a r y route f o r excretion of the toxicant (p.138) make i t tempting to speculate that an i n t e r r e l a t i o n s h i p e x i s t s . F u j i y a (1961, 1965) observed n e c r o t i c changes i n many of the major organs, e s p e c i a l l y the l i v e r , p a r t i c u l a r l y around the b i l i a r y ducts, of f i s h taken from waters re c e i v i n g k r a f t pulp m i l l waste. Tomiyama (1965) proposed that these h i s t o p a t h o l o g i c a l changes may have been caused by r e s i n acids. Although h i s t o p a t h o l o g i c a l observations were not conducted i n the present study, DHA accumulation may r e s u l t i n necrotic changes i n the l i v e r which could account f o r the observed obstructive jaundice. On the other hand, the accumulation of DHA may be secondary to the obstructive jaundice. B i l e flow i s an important determinant of the rate at which many xenobiotics (foreign compounds) are cleared from the plasma and excreted i n the b i l e (Tuttle and Sch o t t e l i u s , 1969). The f a i l u r e or over-loading of the he p a t o b i l i a r y system i n f i s h exposed to DHA may have contributed 165 to the accumulation of t h i s t o x i c a n t i n various organs. This could be e s p e c i a l l y marked i f the b i l e leaked out i n t o the l i v e r parenchyma leading to n e c r o t i c changes and a reduction i n p h y s i o l o g i c a l function. A.reduction i n excretion of DHA v i a the b i l e could compromise the function of other organs. The DHA-induced jaundice may have other chronic effects, r e l a t e d to the d i r e c t i n f l u e n c e of free b i l i r u b i n on t i s s u e metabolism. At elevated plasma b i l i r u b i n l e v e l s such as occur i n i n f a n t s during neonatal jaundice, b i l i r u b i n crosses the blood/brain b a r r i e r and accumulates i n b r a i n t i s s u e . At these l e v e l s , in_ v i t r o studies have demonstrated the uncoupling of oxidative phosphorylation i n i s o l a t e d mitochondria (Gray, 1961).-,These findings are remarkably c l o s e , a l b e i t i n d i r e c t l y , to one of the p o s s i b l e modes of t o x i c a c t i o n of k r a f t m i l l e f f l u e n t on f i s h , as proposed by Warner (1965) . Although these mechanisms remain speculative, hyperbilirubinemia i s frequently r e l a t e d to a reduced clearance due to hypoxia and can i n t e r f e r e with the c e l l volume regulatory mechanism of red blood c e l l s (Wintrobe, 1961). In the present study, increased hematocrit and jaundice have been shown to be c h a r a c t e r i s t i c symptoms of sublethal DHA t o x i c i t y to sockeye salmon. As one of the suggested mechanisms i s one of toxicant-induced hypoxic s t r e s s , there may be a r e l a t i o n s h i p between hypoxia, hematocrit, and the accumulation of b i l i r u b i n i n the blood of salmon exposed to DHA. 166 APPENDIX IV. THE EFFECTS OF SUB-LETHAL DHA EXPOSURE IN FRESH WATER ON THE RED BLOOD CELL DIMENSIONS OF SOCKEYE SALMON SMOLTS In the present study, the sublethal exposure of j u v e n i l e sockeye salmon to DHA i n fresh water i n v a r i a b l y r e s u l t e d i n elevated blood hematocrit values. As a r i s e i n hematocrit may r e s u l t from an increase i n red blood c e l l s i z e , measurements were made of the length and width of erythrocytes taken from exposed and c o n t r o l f i s h . MATERIALS AND METHODS Blood smears were made during blood sampling at the conclusion of the exposure period i n the DHA residue experiment (Appendix 1-4). Duplicate smears were a i r d r i e d , f i x e d i n methyl alcohol and stained with Giesma (Hesser, 1960). The length and width of 25 red blood c e l l s was determined on each s l i d e using an ocular micrometer i n a microscope under 400 x magnifi-cation . C e l l areas were c a l c u l a t e d using the formula(length x width x 0.25 7r)and the r a t i o of width/length was used as a measure of c e l l roundness (Murray and Burton, 1979). As preliminary c a l c u l a t i o n s showed no d i f f e r e n c e between r e p l i c a t e s f o r each f i s h , the values were pooled and the means were c a l c u l a t e d on the basis of the measurements of 50 c e l l s per f i s h . Grand means f o r c e l l area and roundness f o r exposed and c o n t r o l f i s h were then compared using Student's t - t e s t . In addition, the osmolality of pooled plasma samples was measured a f t e r the hematocrits had been determined f o r i n d i v i d u a l f i s h . RESULTS AND DISCUSSION The r e s u l t s (Table XXVI) show that DHA-exposed salmon displayed two of the symptoms c h a r a c t e r i s t i c of sublethal exposure to t h i s toxicant i n f r e s h water; a lowering of plasma osmolality and a r i s e i n hematocrit. Elevated hematocrit 167 was accompanied by a highly s i g n i f i c a n t increase i n the area of the red blood c e l l (Table XXVI) but t h i s occurred without any change i n the degree of c e l l roundness. As the observed r i s e i n hematocrit (17.4%) was considerably greater than the measured change i n c e l l area (10.9%), the d i f f e r e n c e i s probably due to a concomitant increase i n c e l l thickness (height). An approximation of the change i n c e l l volume can be calculated by taking the c e l l height to be ^ 4 p (Eddy, 1977) and assuming that the increase i n height was proportionately s i m i l a r to that observed for the length and width dimensions. By applying the formula for a prolate sphere (V=4/3 TT lwh), where 1, w and h r e f e r to the r a d i i of the red blood c e l l , the 3 mean volume f o r the c o n t r o l c e l l s i s 211 ym . I f the c e l l height increased ^10%, the mean volume of the "exposed" c e l l s would be 367 ym^. This represents an 18% increase i n volume and corresponds w e l l to the observed r i s e of 17.4% i n blood hematocrit. Based on the r e s u l t s of t h i s experiment as well as on a more thorough discussion of other mechanisms which could lead to hemoconcentration (p.98 ), i t can be concluded that the sublethal exposure of sockeye salmon to DHA i n fresh water leads to a d i l u t i o n of the blood plasma and to a swelling of red blood c e l l s which contributes to an increase i n blood hematocrit. Table XXVI. Red blood c e l l dimensions i n fresh water. 3" of j u v e n i l e sockeye salmon exposed to 0.65 mg/L DHA f o r 120 h Length ym Erythrocyte Width Area ym ym2 Blood Plasma Roundness Hematocrit Osmolality % mOsM/kg Exposed 17.02 a±0.08 9.36 b±0.06 125.03 a±0.94 0.552 ±0.005 48.98 ±1.55 282 Control 16.06 ±0.52 8.94 ±0.10 112.74 ±1.82 0.556 ±0.005 41.72 ±0.69 290 Based on the means o f measurements of 50 red blood c e l l s f o r each of 10 f i s h . 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