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Some aspects of the interrelationship of bacterial kidney disease infection and sodium pentachlorophenate… Iwama, George Katsushi 1977

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SOME ASPECTS OF THE INTERRELATIONSHIP OF BACTERIAL KIDNEY DISEASE INFECTION AND SODIUM PENTACHLOROPHENATE EXPOSURE IN JUVENILE CHINOOK SALMON (ONCORHYNCHUS TSHAWYTSCHA) by GEORGE KATSUSHI IWAMA B.Sc, University of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1977 © George Katsushi Iwama, 1977 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements f o r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head o f my Department o r by h is representa t ives . It is understood that copying o r pub l i ca t ion o f th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Z o o l o g y Department or ' The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e A p r i l 15, 1977 ABSTRACT The i n t e r r e l a t i o n s h i p of b a c t e r i a l kidney disease, a chronic disease of cultured salmonids, and an environ-mental toxicant, sodium pentachlorophenate, i n juvenile chinook salmon, over time, was studied. This was carried out by monitoring various haematological parameters i n the f i s h exposed to the two factors, s i n g u l a r l y and i n combination. An evaluation of these parameters as useful indices of s t r e s s -f u l states imposed by the treatment was also attempted. Fur-thermore, external and i n t e r n a l physical examinations, gram stained kidney smears and the occurrence of m o r t a l i t i e s sup-plemented the blood changes i n evaluating the response of the f i s h to these f a c t o r s . Approximately 41 x 10^  viable kidney disease bac-t e r i a , i s o l a t e d from moribund adult pink salmon, were i n -jected i n t r a p e r i t o n e a l l y into the f i s h a f t e r anaesthetiza-t i o n with neutralized tricane methanesulphonate. Control f i s h were s i m i l a r l y sham injected. A l l inje c t i o n s were car-r i e d out on the same day. Both experimental and control groups of f i s h were then exposed to clean water, intermediate and high l e v e l s of sodium pentachlorophenate based on the i n c i p i e n t 96 h L C ^ Q value. The three l e v e l s were: 0 x 96 h L C ^ Q , 0.05 x 96 h L C ^ Q and 0.50 x 96 h L C ^ Q respectively. Four days a f t e r the beginning of toxicant exposure, haematocrit, haemoglobin, red and t o t a l white blood c e l l counts, mean c e l l volume, mean corpuscular haemoglobin concentration, mean c e l l u l a r haemoglobin, blood urea nitrogen, t o t a l protein and plasma glucose values were determined f o r experimental and control groups of f i s h at each toxicant l e v e l . Subsequently, these determinations were made f o r each group of f i s h every four days. This was carried out f o r 3° days a f t e r beginning toxicant exposure unless m o r t a l i t i e s due to the treatments prevented sampling. Pooled (two f i s h per measurement; 12 measurements per sample) blood samples from the severed caudal peduncle were used f o r these determinations. A de-s c r i p t i v e code was developed to categorize the progression of the b a c t e r i a l i n f e c t i o n based on the symptoms of t h i s disease. This code was also used to qu a n t i t a t i v e l y compare' the physical condition of each sample f i s h among the three toxicant l e v e l s . Generally, a synergistic effect was observed be-tween the b a c t e r i a l i n f e c t i o n and toxicant exposure i n the measured blood parameters, physical c h a r a c t e r i s t i c s and the occurrence of m o r t a l i t i e s . This synergistic effect was i n -dicated i n the measured blood parameters (blood urea n i t r o -gen, mean c e l l volume and white blood c e l l count) by an e a r l i e r deviation of the infected f i s h value from the con-t r o l f i s h value with toxicant exposure, an increase i n the difference between infected and control f i s h values with toxicant exposure, or both at any one time or f o r the entire sampling period. The advanced state of physical d e b i l i t a t i o n at both l e v e l s of toxicant exposed infected f i s h r e l a t i v e to the respective control f i s h also indicated synergism between the two fa c t o r s . Furthermore, a catastrophic mortality occurred 0 i n the infected f i s h at the high toxicant l e v e l on the second sampling day that was interpreted as a resu l t of a synergistic effect of the pathogen and the toxicant. In response to the i n f e c t i o n , depressed haema-t o c r i t , haemoglobin, red blood c e l l count, blood urea nitrogen, t o t a l protein and glucose values were observed i n infected f i s h r e l a t i v e to control f i s h over the sampling period. Haemodilution due to pathogenic destruction of osmo-regulatory t i s s u e s was considered the primary cause f o r the observed r e s u l t s . I n h i b i t i o n of erythropoiesis, leakage of proteins through open le s i o n s , depletion of glycogen stores by the multiplying bacteria and cessation of feeding were also considered as addit i o n a l factors that may have c o n t r i -buted to these r e s u l t s . Mean c e l l volume was observed to increase as a res u l t of the i n f e c t i o n . Erythrocytic swelling and i n h i b i -t i o n of erythropoiesis, r e s u l t i n g i n fewer, smaller immature c e l l s , were considered as the causal factors f o r t h i s r e s u l t . Total white blood c e l l counts i n infected f i s h were i n i t i a l l y lower than respective control f i s h but showed an increasing trend with time. This res u l t was seen as an i n i t i a l streee-mediated leucopenia followed by a neutrophilia i n response to the increase i n tissue damage as a resu l t of the disease progression. In response to the toxicant exposure uninfected control f i s h showed decreased haematocrits at the high l e v e l of toxicant exposure. Elevated blood urea nitrogen and glucose values were observed i n the intermediate l e v e l of V toxicant exposed uninfected f i s h r e l a t i v e to the uninfected controls i n clean water. The increased glucose values were interpreted as being caused by an increase i n the secretion of "stress hormones" as a general stress response. These res u l t s l e d to the conclusion that sodium pentachlorophenate exposure reduced the resistance of the f i s h to the e f f e c t s of the kidney disease b a c t e r i a l i n f e c t i o n . It was also con-cluded that some of the measured blood parameters are s e n s i -t i v e indicators of s t r e s s f u l states caused by these f a c t o r s . Haematocrit, red blood c e l l count, mean c e l l v o l -ume and t o t a l / d i f f e r e n t i a l white blood c e l l count measure-ments are recommended fo r routine monitoring of the physio-l o g i c a l conditions of f i s h stocks f o r the purposes of stress detection. The careful evaluation of'the physiological 1 con-d i t i o n of f i s h stocks i s recommended as a part of bioassay procedures f o r the purpose of making meaningful comparisons between te s t r e s u l t s . v i ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to Dr. G.L. Greer and Dr. D.J. Randall f o r t h e i r patient supervision and stimulating guidance through t h i s study. Their valuable advice i n the preparation of t h i s manuscript i s greatly appreciated. I would also l i k e to thank Dr. G. B e l l , Dr. J.C. Davis and Mr. H. Sparrow fo r t h e i r h e l p f u l suggestions and p r o f i t a b l e discussions of various aspects of t h i s i n v e s t i g a t i o n . The advice of Dr. P.A. Larkin con-cerning the s t a t i s t i c a l treatment of my data i s also appreciated. I am indebted to Dr. T. Evelyn, and his assistants for t h e i r valuable advice and patient guidance concerning the microbiological aspects of t h i s study. I would also l i k e to thank Ms. Margaret Fisher f o r her assistance i n the preparation and the typing of t h i s report. F i n a l l y , I would l i k e to thank a l l the people at the P a c i f i c Environment I n s t i t u t e Who d i r e c t l y or i n d i r e c t l y contributed to t h i s study i n t h e i r various c a p a c i t i e s . v i i TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGMENTS v i TABLE OF CONTENTS v i i LIST OF TABLES i x LIST OF APPENDIX TABLES x LIST OF FIGURES x i LIST OF APPENDIX FIGURES x i i i SECTION I - INTRODUCTION 1 SECTION II - INITIAL EXPERIMENTS I n i t i a l Experiment A - Infection'Experiment Introduction 7 Material and methods # Results 9 Discussion 12 I n i t i a l Experiment B - Bioassay Introduction 14 Material and methods 15 Results and discussion 16 SECTION III - MATERIALS AND METHODS Experimental design.... 2 0 Fish 2 0 Acclimatization 2 0 Apparatus 23 Infection of f i s h 23 Toxicant administration 24 Sampling.... 26 Parameter measurement 27 Pathological examination 28 Data analysis 2 9 SECTION IV - RESULTS Comparison between uninfected control f i s h and kidney disease uninfected f i s h f o r a l l t o x i -cant l e v e l s 32 Comparison among uninfected control f i s h group fo r a l l toxicant l e v e l s 36 Comparison among kidney disease infected f i s h f o r a l l toxicant levels.. 38 SECTION V - DISCUSSION 57 SECTION VI - GENERAL CONCLUSIONS AND RECOMMENDATIONS 6 7 SECTION VII - BIBLIOGRAPHY 70 v i i i SECTION VIII - APPENDICES Appendix I - Sodium pentachlorophenate Preparation of stock solution of NaPCP... Preparation of bioassay solutions i n modified Mariot bottles Preparation of toxicant solutions f o r the main experiment Appendix II - Microbiological Procedures Preparation of Evelyn's kidney disease I s o l a t i o n and growth of kidney disease bacteria , Harvesting of kidney disease b a c t e r i a l cells.and preparation of inoculum for i n j e c t i o n into f i s h Viable counts Appendix III - Categorization of physical c h a r a c t e r i s t i c s i n response to kidney disease i n f e c t i o n Appendix IV - Tables of mean, standard error of the mean and grand mean values f o r HCT, Hb, RBC, WBC, MCV, BUN, TP and GLU.. Appendix V - Results for mean corpuscular haemoglobin concentration and mean c e l l haemoglobin Page 77 77 77 79 80 82 S3 85 88 97 Appendix VI - Photographs of apparatus used i n bioassay and the main experiment... 105 i x LIST OF TABLES Page TABLE I Incubation times r e s u l t i n g from d i f f -erent concentrations of kidney disease bacteria injected i n t r a p e r i t o n e a l l y into juvenile coho salmon 10 TABLE II Symptoms of b a c t e r i a l kidney disease 10 TABLE III Physical c h a r a c t e r i s t i c s code and mor-t a l i t y f o r the main experiment 39 TABLE IV Comparison of various haematological values measured i n t h i s study with those of Thomas et. a l . (1969) 56 X LIST OF APPENDIX TABLES Page Appendix VI Tables of mean,standard error of the mean and grand mean values. TABLE I Haematocrit 88 TABLE II Haemoglobin 89 TABLE III Red blood c e l l count 90 TABLE IV Total white blood c e l l count 91 TABLE V Mean c e l l volume 92 TABLE VI Blood urea nitrogen 93 TABLE VII Total protein 94 TABLE VIII Glucose 95 Appendix V Tables of mean, standard error of the mean and grand mean values. TABLE I Mean corpuscular haemoglobin concentration 99 TABLE II Mean c e l l u l a r haemoglobin 100 x i LIST OF FIGURES Page FIGURE l a . Photograph of bioassay aquaria as described i n I n i t i a l Experiment B 18 FIGURE l b . Diagrammatic representation of one bioassay aquarium.. 18 FIGURE II T o x i c i t y curve showing L T ^ Q ' S 19 FIGURE III F a c t o r i a l design used f o r the main experiment 21 FIGURE IVa. Photograph of a p a i r of te s t tanks used i n the main experiment 22 FIGURE IVb. Diagrammatic representation of the same tanks from the back 22 FIGURES V to XII Graphs of the responses of measured haematological parameters i n the d i f f -erent groups of f i s h to the experimental treatments, b a c t e r i a l kidney disease i n -fe c t i o n and NaPCP exposure, over the sam-pl i n g period. FIGURE V Haematocrit i 40 FIGURE VI Haemoglobin 41 FIGURE VII Red blood c e l l count 42 FIGURE VIII Total white blood c e l l count 43 FIGURE IX Mean c e l l volume 44 FIGURE X Blood urea nitrogen 45 FIGURE XI Total protein 46 FIGURE XII Glucose 47 FIGURES XIII to XX Histograms showing grand means and means of absolute differences between control and experimental f i s h f o r the d i f f e r e n t groups of f i s h . FIGURE XIII Haematocrit FIGURE XIV Haemoglobin 48 49 x i i LIST OF FIGURES (cont'd) Page FIGURE XV Red blood c e l l count .50 FIGURE XVI White blood c e l l count 51 FIGURE XVII Mean c e l l volume 52 FIGURE XVIII Blood urea nitrogen 53 FIGURE XIX Total protein 5k FIGURE XX Glucose 55 x i i i LIST OF APPENDIX FIGURES Page Appendix V Graphs of the response of MCHC and MCH values i n the di f f e r e n t groups of f i s h to the experimental t r e a t -ments. FIGURE I Mean corpuscular haemoglobin concentration 1 0 1 FIGURE II Mean c e l l u l a r haemoglobin 1 0 2 FIGURE III Mean corpuscular haemoglobin concentration..... 1 0 3 FIGURE IV Mean c e l l u l a r haemoglobin 1 0 4 1a SECTION I INTRODUCTION 1 A l l l i v i n g organisms are subject to perturbations i n t h e i r environment which can lead to deviations from t h e i r normal state. The perturbations can be i n the form of phy-s i c a l , chemical or b i o l o g i c a l factors and be both i n t e r n a l and external to the organism. Some deviation from a normal state at any given time i s i n i t s e l f a normal event and represents the adaptive responses which t y p i c a l l y function to o f f s e t the eff e c t of perturbations, eg. homeostasis. Consequently, merely the presence of some environmental factor(s) does not mean an abnormal or even pathological state w i l l ensue unless the factors cause the normal range of adaptive responses to be exceeded. Stress, as defined by Brett ( 1 9 5 8 ) , w i l l be used i n t h i s study to mean: "...a state produced by an environ-mental or other factor which extends the adaptive responses of an animal beyond the normal range or which disturbs the normal functioning to such an extent that i n either case, the chances of su r v i v a l are s i g n i f i c a n t l y reduced." Whether i t be an environmental stressor lowering the resistance of the organism to a b i o l o g i c a l pathogen or a pathogenic stressor lowering the resistance of the organism to the noxious e f f e c t s of an environmental toxicant, the physio-l o g i c a l i n t e r a c t i o n that takes place between the organism and one or more of these stressing agents may be important to i t s s u r v i v a l . 2 In discussing the role of stress i n the disease resistance i n f i s h , Wedemeyer (1974) stated: "Stress r e -quiring adjustments that exceed a f i s h ' s a b i l i t y to accom-modate w i l l be l e t h a l . Less severe stress w i l l predispose to p h y s i o l o g i c a l disorders, or to infectious diseases i f f i s h pathogens are present." Furthermore, writing on the disease mechanisms i n crustaceans and marine arthropods, Bang (1970) commented, "Since the time of Metchnikoff i t has been recognized that disease mechanisms are a part of the biology of any organism, and that pathology i s related to physiology. ...As a re s u l t of the selection force of diseases, mechanisms develop to r e s i s t disease. The i n t e r -action between such destructive forces and the host's r e -sponses to them i s disease." The recognition of t h i s i n t e r a c t i o n i s becoming increasingly evident i n the l i t e r -ature. Writing on disease resistance i n f i s h e s , Wedemeyer (1974) supported t h i s view that diseases are not single cause events but are the end r e s u l t of the i n t e r a c t i o n of the disease-causing agent, the f i s h and the environment. A v a r i e t y of pathogens may be present i n the water but the f i s h may not become diseased u n t i l a stressing agent reduces the defense system of the f i s h beyond some c r i t i c a l point. Cultured fishes are often subjected to a v a r i e t y of s t r e s s f u l conditions: excessive handling, crowding, f l u c t u a t i n g water conditions and drug treatments. Under such conditions, a number of the diseases encountered may be stress-mediated. A number of s p e c i f i c diseases have been correlated with the presence of certain environmental 3 conditions (Wedemeyer, 1970, 1974). Epizootics also occur i n wild stocks of f i s h and t h e i r coincidence with unusual environmental changes i s well documented i n f i s h e r i e s l i t e r a t u r e . For example, Snieszko (1974) reviewed the coincidence of infectious diseases with environmental stress caused by temperature, eutrophication, sewage, metabolic products of f i s h , i n d u s t r i a l p o l l u t i o n and p e s t i c i d e s . In t h i s context, he stated: "It i s well known, from epidemology, that an infectious agent causes a disease of the host i f environmental conditions are r i g h t . The influence of each subset i s variable - disease breaks out only i f there i s a s u f f i c i e n t r e l a t i o n s h i p between them... I f the occurrence of stress coincides with the presence of pathogenic micro-organisms, i t i s l o g i c a l to assume that outbreaks of disease are more l i k e l y to take place." Recognizing the importance of the role of stress due to a b i o t i c factors i n disease outbreaks i n f i s h , i t would be desirable to be able to detect the s t r e s s f u l state before the diseased state i s reached and an outbreak occurs. The increasing volume of l i t e r a t u r e reporting physiological responses of stress i n f i s h r e f l e c t s the growing awareness by investigators of the importance of knowing the physio-l o g i c a l condition of experimental, cultured or wild f i s h stocks. It has been reported i n t h i s l i t e r a t u r e that a v a r i e t y of haematological changes occur as a res u l t of the metabolic response of the f i s h to the stressing agent. Some of these blood changes may be indicators of the 4 s t r e s s f u l state i n f i s h and thus are useful i n i n i t i a t i n g prophylactic measures i n f i s h culture and provide some de-gree of assessment of ph y s i o l o g i c a l status of f i s h stocks. The general objective of the present study was to investigate the p o s s i b i l i t y that some haematological charac-t e r i s t i c s of juvenile chinook salmon (Oncorhynchus tshawytscha) might r e f l e c t stress, as defined above. This was to be done by monitoring various blood parameters during exposure to two possible stressing agents, a b a c t e r i a l pathogen and an en-vironmental toxicant, presented si n g l y and i n combination. The blood parameters that were monitored were haematocrit (packed c e l l volume), haemoglobin, red blood c e l l count, t o t a l white blood c e l l count, mean c e l l u l a r haemoglo-bin content, mean corpuscular haemoglobin concentration, mean c e l l volume, t o t a l plasma protein, blood urea nitrogen and plasma glucose. The b i o t i c agent used i n t h i s study was b a c t e r i a l kidney disease. Although acute and subacute forms of t h i s disease occur sporadically, i t i s mainly chronic. It i s also widespread, having been reported i n Europe, North America and Japan. A l l salmonids are considered susceptible to kidney disease but i t seldom occurs i n f i s h l e s s than s i x months o l d . The disease i s characterized i n t e r n a l l y by an enlarged, edema-tous kidney which usually exhibits off-white lesions that vary i n size and number and gives r i s e to the name kidney d i -sease.. The lesions also can occur i n other organs such as the l i v e r , spleen and heart. A turb i d f l u i d i n the abdominal and 5 p e r i c a r d i a l c a v i t i e s i s often symptomatic as w e l l . Externally f i s h i n advanced.stages of the disease usually exhibit a d i s -tended abdomen, exopthalmia, skin petechiation, welts, lesions and tissue decay. No cure f o r t h i s disease i s known at pre-sent The toxicant used i n t h i s study was sodium penta-chlorophenate (NaPCP). I t i s a p e s t i c i d a l wood preservative that i s commonly used i n North America. In agr i c u l t u r e , i t i s used as a defoliant and herbicide while i n industry and households i t i s used f o r t r e a t i n g wood against termites and as a preservative of various products prone to micro-b i a l attacks (Hoben et. a l . , 1976). After screening 35 chemicals on the basis of t h e i r chemical properties, Alderdice (I963) selected NaPCP as a suitable toxicant f o r f i s h research because of i t s known toxic action i n f i s h and better under-standing of i t s chemical behavior compared to many other t o x i -cants. F i n a l l y , NaPCP i s an environmental contaminant which could function as an a b i o t i c stressor to f i s h and therefore i s of p r a c t i c a l interest as w e l l . Alderdice (1963) also reviewed the b i o l o g i c a l action of pentachlorophenol and i t s sodium s a l t , sodium pentachlorophenate (NaPCP). He reported that work on s n a i l tissue and rabbit muscle preparations indicated the toxic action of both pentachlorophenol and NaPCP was by uncoupling oxidative phosphorylation. In addi-t i o n , adenosinetriphosphotase a c t i v i t y and g l y c o l y t i c phos-phorylation are i n h i b i t e d . More recently, several studies reporting on the 6 metabolism o f pentachlorophenol i n f i s h (Kobayashi and Akitake , 1975 a, b , c; Akitake and Kobayashi, 1975; Kobayashi e t . a l . , 1975; Kobayashi e t . a l ., 1975; Kobayashi e t . al.,1975 Kobayashi e_t. a l ., 1976) ind ica te that decomposition and b i l i a r y excretion, a f ter d e t o x i f i c a t i o n by sulphate conjuga-t i o n i n the hepatopancreas, i s the main pathway of e l imina-t i o n of pentachlorophenols i n f i s h . The main features o f the present study can be seen as a ser ies o f more s p e c i f i c object ives which were accomplished as fo l lows: 1) to determine i f several o f measured blood parameters i n juveni le Chinook salmon might r e f l e c t a s t r e s s f u l state im-posed by e i ther the b a c t e r i a l pathogen, the toxicant or both, 2) to character ize both the disease process and the tox ic ef fects o f sodium pentachlorophenate by the measured blood parameters, 3) to determine i f s i g n i f i c a n t change i n these blood para-meters precede overt symptoms of the i n f e c t i o n or response to the tox i can t , thus evaluating the s e n s i t i v i t y o f these parameters as ind ica tor s o f the s t r e s s f u l s ta te , and 4) to invest igate the p o s s i b i l i t y that the stress imposed by the toxicant might reduce the res i s tance of the f i s h to the d e b i l i t a t i v e ef fects o f the pathogen. 7a SECTION II INITIAL EXPERIMENTS 7 INITIAL EXPERIMENT - A INFECTION EXPERIMENT INTRODUCTION This i n i t i a l experiment was carried out to deter-mine the progression of b a c t e r i a l kidney disease f o r d i f f e r -ent concentrations of b a c t e r i a l suspensions harvested from culture media and injected i n t r a p e r i t o n e a l l y . The experi-ment also provided the opportunity to develop a coding system to describe the symptoms a r i s i n g during the progression of the disease f o r the main experiment. Although juvenile chinook salmon were used f o r the subsequent experiments, coho salmon (Oncorhynchus kisutch) were used f o r t h i s i n i t i a l experiment. Coho was the species of choice f o r the entire study because they are cultured int e n s i v e l y i n the P a c i f i c Northwest region and b a c t e r i a l kidney disease i n coho i s a chronic problem i n many f i s h culture operations. Furthermore, the response of several blood parameters to another environmental toxicants, dehydro-i' a b i e t i c acid, has been reported for t h i s species (Iwama et. a l . , 1976) and the present study would provide additional comparison but with a d i f f e r e n t toxicant. The coho held i n reserve f o r the study were, however, l o s t through accident and a replacement stock could not be obtained. Since chinook are cultured quite in t e n s i v e l y as well and susceptible to s i m i l a r c ulturing problems, t h i s species had p r a c t i c a l i n t e r -est and was used to complete the study. The use of chinook was j u s t i f i e d on the basis that the incubation time and general symptoms of the disease appear to be s i m i l a r between 8 different species and sizes of salmonids for a particular concentration of experimentally injected kidney disease bacterial suspension (T. Evelyn - per. comm.). It was therefore judged that the resulting incubation time deter-mined for the coho could be used for the subsequent experi-ment with chinook salmon. MATERIAL AND METHODS  FISH Sixty juvenile coho of length 11.0 + 0.75 cm and weight 14.11 + 2.01 g (mean + S.D.) were transferred from a 7000 1 outdoor holding tank receiving Cypress Creek water, 4 to 6 C, to three 64 1 tanks in the laboratory receiving well water (9-11 C; pH 6.8-6.9; 0 2 8-8.5 mg/1; hardness 54 mg/1 CaC0 3). ACCLIMATIZATION The fish were l e f t for 48 h to acclimatize to the laboratory environment before daily feeding (5/32 Oregon Moist Pellet) was resumed. PROCEDURE The bacterial c e l l suspension for injection into the fish was harvested (App. IIC) from two petri plates of kidney disease bacteria (strain DR151) 15 days after inocu-lation when the cells were in their log-phase of growth (T. Evelyn, per. comm.). The bacteria were grown on ^ Evelyn's kidney disease media.j--j.-j- and obtained from the Micro-biology department at the Pacific Biological Station 9 (Nanaimo, B.C.). Three bacterial suspensions having optical densities of 0.25, 0.10 and 0.01 (420 nm) were prepared (App. IIC) for this experiment. After a 24 h starvation period, and before injec-tions, three groups of 20 fish each were anaesthetized in 100 mg/1 tricane methanesulphonate (MS 222, Kent Lab. Ltd. Vancouver, B.C.) neutralized with 2N NaOH. Each group of fi s h received one of the concentrations of bacterial suspen-sion by intraperitoneal injection through the mid-lateral body wall just anterior to the pelvic f i n . Syringe needles of 27.5 g were used to eliminate loss of inoculum through the needle puncture after the needle was withdrawn. RESULTS Table I summarizes the mortality results of the experiment with juvenile coho and shows the respective incubation times observed with the different concentrations of injected bacterial suspension. These results indicate that a bacterial c e l l suspension of 0.10 O.D. (420 nm) would be the appropriate inoculum concentration for the derived 40 day period for the main experiment (App. IIC). The symptoms of kidney disease observed in fish dying in this experiment are presented in Table II. 10 T a b l e I I n c u b a t i o n t i m e s r e s u l t i n g f r o m d i f f e r e n t c o n c e n -t r a t i o n s o f k i d n e y d i s e a s e b a c t e r i a i n j e c t e d i n t r a -p e r i t o n e a l l y i n t o j u v e n i l e coho s a l m o n . C o n c e n t r a t i o n o f k i d n e y d i s e a s e 0.25 0.10 0.01 b a c t e r i a i n j e c t e d ( o p t i c a l d e n s i t y ) I n c u b a t i o n t i m e ( d a y s ; 34-45 34-49 49-58 f i r s t - l a s t m o r t a l i t y ) P e r c e n t s u c c e s s f u l 100$ 100$ 100$ i n f e c t i o n ( m o r t a l i t y ) T a b l e I I Symptoms o f b a c t e r i a l k i d n e y d i s e a s e O b s e r v e d ( p e r c e n t a g e o f t o t a l m o r t a l -i t y f o r a l l c o n c e n t r a t i o n s ) E x o p t h a l m i a P e t e c h i a e (9096) (40$) W e l t s (4596) R e f e r e n c e s B u l l o c k e t . a l . , 1975; E a r p e t . a l . , 1955; S n i e s z k o and G r i f f i n , 1955; Wood and W a l l i s , 1955 B u l l o c k e t . a l . , 1975; E a r p e t . a l . , 1955; R u c k e r e t . a l . , 1955; S n i e s z k o and G r i f f i n , 1955; Wood and W a l l i s , 1955 B u l l o c k e t . a l . , 1975; B e l l , 1961; S n i e s z k o and G r i f f i n , 1955; Wood and W a l l i s , 1955 S n o u t / U p p e r Jaw Decay (75$) T h i s s t u d y Y e l l o w f l u i d f r o m v e n t (30$) D i s t e n d e d abdomen (100$) S n i e s z k o and G r i f f i n , 1955; Wood and W a l l i s , 1955 Wood and W a l l i s , 1955; S n i e s z k o and G r i f f i n , 1955; B e l l , 196l 11 Overall dark colouring (95%) Haemorrhaging vent (50$) Haemorrhaging i n major organs and body cavity walls (85$) Excessive f l u i d i n body cavity (100$) Enlarged, pale spleen (30$) •Smokey' appearance of swim bladder Lesions on heart, l i v e r , spleen and blood vessels (95$) F r i a b l e , pale l i v e r t issue (45$) Empty alimentary canal (100$) Distended hind gut (65$) Swollen pale kidney-lumps (lesions) (100$) Gram p o s i t i v e rods ( D i p l o b a c i l l i ) i n kidney-smears > (100$) Earp et. a l . , 1955 Earp et. a l . , 1955 Bullock et. a l . , 1975 Bullock et. a l . , 1975; Earp et. a l , 1955; Rucker et. a l . , 1955; Wood and W a l l i s , 1955 Bullock et. a l . , 1975 (50$) This study B e l l , 1961; Earp et. a l . , 1955; Snieszko and G r i f f i n , 1955; Wood and W a l l i s , 1955 This study This study Bullock et. a l . , 1975 Bullock et. a l . , 1975; B e l l , 1961; G r i f f i n , 1954; Earp et. a l . , 1955; Rucker et. a l . , 1954; Bendele and Klontz, 1975; Snieszko and G r i f f i n , 1955; Wood and Wa l l i s , 1965; Rucker et. a l . , 1951 A l l references f o r previous symptoms. 12 DISCUSSION The incubation times observed i n t h i s experiment were found to be i n general agreement with Evelyn et. a l . ( 1 9 7 3 ) . In that study, incubation times of 22 to 36 days and 17 to 31 days were observed i n 86 g (mean) sockeye salmon (Oncorhynchus nerka) s i m i l a r l y injected at opacities of 1 . 0 and 1 0 . 0 OD at 420 nm respectively. A proportionally longer incubation time of 34 to 49 days resulted from a ten-fold lower concentration, 0 . 1 0 OD, of b a c t e r i a l c e l l s injected i n t h i s study. The majority of the observed symptoms of the disease are i n agreement with those reported i n the l i t e r a -ture, as seen i n Table I I . The symptoms suggest that t h i s disease i s a systemic i n f e c t i o n that a f f e c t s a l l parts of the host's body. A l l the reported symptoms related to t h i s disease were observed i n t h i s experiment except the formation of white, f a l s e membranes (Earp et. a l . , 1955)• It was thought, however, that the "smokey" appearance of the swim-bladder, previously unreported, may i n f a c t have been the f a l s e membranes. Upon microscopic examination of the surface of the swimbladders, small, white, pinpoint lesions were observed l i n i n g a l l the blood vessels, thus giving a "smokey" appearance. This suggests that the bacteria spreads through-out the body v i a the c i r c u l a t o r y system. Earp et. a l . (1955) stated that the bacterium of kidney disease could be found r e a d i l y i n the c i r c u l a t o r y system of f i s h i n advanced stages of the disease. This would also explain the observation that the majority of lesions were found i n organs that are highly 13 vascularized (eg. kidney, l i v e r and spleen). Furthermore, petechiation and haemorrhaging at the various s i t e s indicate the destruction of vascular t i s s u e of the c i r c u l a t o r y system by the pathogen. Destruction of the renal tissue i s also a primary c h a r a c t e r i s t i c of t h i s disease (Wood and Yasutake, 1 9 5 6 ) . Therefore, f i s h i n fresh water that osmotically imbibe water from t h e i r hypotonic environment would gain more water as t h e i r excretory a b i l i t i e s decrease. This e f -fect explains the excessive f l u i d i n the abdominal cavity and the distended abdomen of infected f i s h . The exopthalmia r e s u l t i n g from t h i s i n f e c t i o n may have been caused by the pressure exerted from the increasing f l u i d volume 'in the body ..as suggested by Wood and Wallis ( 1 9 5 5 ) . The o v e r a l l dark colouring, or melanosis, that was observed i n m o r t a l i t i e s and moribund f i s h was interpreted as being a non-specific hormonal or nervous response to abnormal conditions i n i t s i n t e r n a l or external environment. This effect has been observed i n f i s h of the same species and age i n response to toxicant exposure and high l e v e l s of exercise (unpublished observations). 14 INITIAL EXPERIMENT - B BIOASSAY INTRODUCTION A bioassay was conducted to determine the 96 h L C ^ Q of NaPCP under a s i m i l a r f i s h loading density that would be used i n the main experiment. Since at t h i s point i n the study, the coho had been l o s t , t h i s experiment had additional importance because the t o x i c i t y of NaPCP f o r juvenile chinook salmon has not been reported. FISH Juvenile chinook salmon of length 1 0 . 3 + 0.8 cm and weight 1 1 . 2 5 + 3 . 9 4 g (mean + S.D.) were reared i n a 4000 1 c i r c u l a r tank indoors under natural photoperiod i n well water ( 1 0 - 1 1 C; pH 6.8 - 6 . 9 ; 0 2 8-8.6 mg/1; hardness 54 mg/l CaCO^). Gram s t a i n of kidney smears a s e p t i c a l l y taken from a random sample indicated that t h i s stock was free of b a c t e r i a l pathogens. APPARATUS Six 3 2 . 6 1 glass aquaria set i n one large rectan-gular f i b e r g l a s s tank were used as te s t tanks f o r the flow-through bioassays of NaPCP. The large rectangular tank provided some degree of i n s u l a t i o n to diurnal temperature fluctuations by c o l l e c t i n g the overflow from the test aquaria and maintaining a water depth equal to approximately 2/3 the aquaria height. The four sides of each aquaria were covered with black p l a s t i c and the top covered with smoked 15 plexiglas to reduce external s t i m u l i . An enclosed funnel connected to a glass tube entering the dquarium through a hole i n the plexiglas top mixed and introduced the toxicant and diluent water before i t was added to each te s t tank (App. VI; F i g . I ) . The stock solutions of NaPCP were prepared as described by Alderdice (1963; see also App. IA). Diluent water entered the mixing funnels at a constant flow of 880 ml/min through 5 mm diameter polyethylene tubing. The stock solution of NaPCP flowed to the mixing funnels from 25 1 modified Mariot bottles (Leduc, 1966) at a constant rate of 3.9 ml/min through 2 mm, outside diameter, polyethylene tubing ( F i g . I ) . The diluent water flow provided a 90.0 percent replacement time of approximately 1.5 h (Sprague, 1969). PROCEDURE The concentrations of NaPCP used i n the bioassay were 0.06, 0.07, 0.08, 0.09, 0.13, 0.19 and 0.27 mg/1. Each bioassay tank contained 40 f i s h r e s u l t i n g i n a loading den-s i t y of approximately 13.8 g / l ; the approximate loading den-s i t y that would be used i n the main experiment. Water condi-tions i n the t e s t aquaria were maintained at 11.8-12.0 C; pH 7.0-7.1 and 0 2 8-8.3 mg/1. After the toxicant flows were started, the concentrations i n the t e s t tanks were allowed to e q u i l i b r a t e f o r 4 h, which was the time, f o r 99.9 percent r e -placement at the diluent flows used (Sprague, 1968). At the end of t h i s e q u i l i b r a t i o n period, observations f o r mortality were 16 made at 0 . 2 5 , 0 . 5 , 1 . 0 , 2 . 0 , 4 . 0 h and every 2 + 0 . 5 h thereafter. After the f i r s t mortality occurred, observa-t i o n s were made at more frequent i n t e r v a l s . Death was recorded as the time when opercular movements stopped. Excess water was removed from dead f i s h with paper tissue before obtaining the wet weight. DETERMINATION OF THE 9 6 h LCg Q The median s u r v i v a l time (L T ^ Q) was obtained from log-probit plots of cumulative percent mortality (Sprague, 1 9 6 9 ) . These data were used to construct a t o x i -c i t y curve by p l o t t i n g log-median s u r v i v a l times against l o g -toxicant concentration ( F i g . I I ) . The i n c i p i e n t l e t h a l con-centration (LCJJQ) was determined from a log-probit plot of percentage dead at 96 h (probit scale) against concentration (log scale)(Sprague, 1 9 6 9 ) . Following the nomographic proce-dure of L i t c h f i e l d and Wilcoxon ( 1949) a l i n e best f i t t i n g the points was drawn by minimizing the Chi value of the l i n e . This procedure y i e l d s a r e l a t i v e l y accurate estimate of the i n c i p i e n t L C ^ Q value and also allows the c a l c u l a t i o n of the 9 5 percent confidence l i m i t s and a slope function, s. The slope function permits reproduction of the l i n e . RESULTS AND DISCUSSION The i n c i p i e n t 9 6 h LC^ Q of NaPCP f o r juvenile chinook salmon at a loading density of approximately 1 3 . 8 g/1 was 0 . 0 7 8 mg/1. The 9 5 percent confidence l i m i t s were 0 . 1 1 and 0 . 0 5 7 mg/1. The f i s h i n the control tank were observed 1 7 to be normal i n behavioral and physical c h a r a c t e r i s t i c s at the end of the bioassay. The r e l a t i v e l y high loading den-s i t y did not seem to have any obvious adverse effects on the control f i s h . The 9 6 h L C ^ Q estimation i n t h i s study approximates those f o r other salmonids. 9 6 h L C ^ Q values of NaPCP f o r juvenile rainbow trout (Salmo gai r d n e r i ) , coho salmon (Oncorhynchus kisutch) and sockeye salmon (Oncorhynchus  nerka) have been reported as 0 . 0 9 8 mg /1, 0 . 0 9 2 mg/1 and 0 . 1 3 0 mg/1 respectively by Davis and Hoos ( 1 9 7 5 ) . As recommended by the guidelines f o r conducting bioassays (Sprague, 1 9 6 9 ; Standard Methods, 1 9 7 1 ; Davis and Mason, 1 9 7 3 ) , a low loading density of 0 . 5 mg/1 was used i n the s t a t i c bioassays involved i n the above study (Davis and Hoos, 1 9 7 5 ) . The s i m i l a r i t y between the r e s u l t s of the present experiment and those reported by Davis and Hoos ( 1 9 7 5 ) suggests that carrying out a bioassay under flow through conditions with a r e l a t i v e l y high toxicant solution replacement rate may ameliorate the eff e c t s of using a high loading density. The possible stress that may be imposed on the f i s h by a high loading density would otherwise be expected to s i g n i f i c a n t l y lower the 9 6 h L C J J Q value by reducing the resistance of the f i s h to the eff e c t s of the toxicant. n 18a P h o t o g r a p h o f b i o a s s a y a q u a r i a as d e s c r i b e d i n I n i t i a l E x p e r i m e n t B. D i a g r a m m a t i c r e p r e s e n t a t i o n o f one b i o -a s s a y a q u a r i u m s h o w i n g e n c l o s e d m i x i n g f u n n e l , t e s t t a n k and m o d i f i e d M a r i o t b o t t l e c o n t a i n i n g t o x i c a n t . 18 F i g u r e I b. m o d i f i e d M a r i o t b o t t l e c o n t a i n i n g t o x i c a n t 19a Figure II T o x i c i t y curve showing L T ^ Q ' S . the i n c i p i e n t 96 h L C ^ Q i s indicated with 95$ confidence l i m i t s as determined by nomographic a n a l y s i s . 19 96 h (5760 min) E-1 E-< M < EH Pi O O i r \ O EH I I — 1 . E-i i n c i p i e n t 96 h L C ^ Q + 95$ confidence l i m i t s = -0.07.8 mg/1 (0.057, 0.11) NaPCP CONCENTRATION - mg/1 Figure I I 20a SECTION III MATERIALS AND METHODS FOR MAIN EXPERIMENT V 20 EXPERIMENTAL DESIGN A two x three x nine f a c t o r i a l design was used f o r t h i s experiment ( F i g . I I I ) . The two l e v e l s of the health fac t o r were uninfected control f i s h and kidney disease i n -fected experimental f i s h . The three l e v e l s of the environ-mental condition factor were 0 , 0 . 0 5 and 0 . 5 0 of the i n c i p -ient 9 6 h L C ^ Q value f o r the toxicant NaPCP as determined i n I n i t i a l Experiment B. The t h i r d factor was time, with nine sampling days at four-day i n t e r v a l s . FISH Juvenile chinook salmon of length 1 1 . 5 + 0 . 4 cm and weight 1 5 . 6 2 + 1 . 9 9 g (mean + S.D.) were obtained from Rosewall Creek on Vancouver Island where they had been reared from f r y . The eggs were from wild stock returning to the Qualicum Salmon Hatchery on Vancouver Island. The f i s h were transferred by truck to the P a c i f i c Environment I n s t i t u t e and 2 4 0 f i s h introduced d i r e c t l y into each of s i x tes t tanks receiving well water ( 1 2 - 1 2 . 2 C; 0 2 8 - 8 . 5 mg/1; pH 6 . 5 - 6 . 9 ; hardness 5 4 mg/1 CaCO^). ACCLIMATIZATION The f i s h were acclimated to the experimental tanks f o r 2 7 days before experimental treatment. They were fed a commercial f i s h food ( 5 / 3 2 " Oregon Moist P e l l e t ) twice d a i l y to s a t i a t i o n . To condition the f i s h to the presence of the sampling net, the f i s h were fed only a f t e r the tank covers were l i f t e d and the sampling net introduced into the water. It was thought that t h i s procedure would reduce the time f o r 21a F i g u r e I I I F a c t o r i a l d e s i g n u s e d f o r t h e m a i n e x p e r i m e n t . F a c t o r s a r e : H e a l t h (C = u n i n f e c t e d c o n t r o l f i s h ; E = k i d n e y d i s e a s e i n f e c t e d e x p e r i m e n t a l f i s h ) ; E n v i r o n -m e n t a l C o n d i t i o n (1 = c l e a n w a t e r , 0 x 96 h L C ^ Q ; 2 = i n t e r m e d i a t e l e v e l o f NaPCP e x p o s u r e , 0.05 x 96 h L C ^ Q 3 = h i g h l e v e l o f NaPCP e x p o s u r e , 0.5 x 96 h L C ^ Q ) ; Time ( T I = f i r s t s a m p l i n g d a y , f o u r days a f t e r b e g i n -n i n g t o x i c a n t e x p o s u r e ; 2 - 9 = s u b s e q u e n t s a m p l i n g days a t f o u r day i n t e r v a l s ) . 21 Figure III EXPERIMENTAL DESIGN HEALTH IC IE o H E—' M Q o o \ N 22a F i g u r e IV a . P h o t o g r a p h o f a p a i r o f t e s t t a n k s u s e d i n main e x p e r i m e n t . b . D i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e same t a n k s f r o m t h e b a c k . T o x i c a n t r e s e r -v o i r s , t o x i c a n t h e a d t a n k , e n c l o s e d m i x i n g f u n n e l s and d i l u e n t p i p e s a r e shown. 2 2 Figure IV a. b . d i luent water from constant head tank enclosed mixing funnel 23 netting the f i s h , thereby reducing the disturbance when reg-ular sampling began. Oxygen, temperature and pH measure-ments for water in each tank were taken in the morning before the f i r s t feeding and at intervals of three to four days during this acclimatization and conditioning period. APPARATUS To provide aerated water at a constant flow to the test tanks, well water (10-11 C; pH 6.8-6.9; 0 2 8-8.5 mg/1; hardness 54 mg/1 CaCO^) was passed through six aspir-ators into a 186 1 fiberglas head tank. Additional aeration was provided with compressed a i r . The flow of diluent water into the mixing funnels located on each of the six 186 1 test tanks was regulated independently by valves. The apparatus for administering a l l the levels of NaPCP were the same. The toxicant was pumped from 100 1 plastic reservoirs into 10 1 plexiglas head tanks and the overflow returned to the reservoirs. From the toxicant head tanks, a constant flow of 40 ml/min was led through 5 mm plastic tubing into the mixing funnels (Fig. IV). Diluent water flow was 3.2 l/min and provided a 99.9 percent replace-ment time of approximately 4.5 h as determined by the graph-i c a l method of Sprague (1969). INFECTION OF FISH As a precaution, uninfected control fish were a l -ways handled before infected fish for a l l procedures through-out the experiment. Before sham injection, control fish in groups of 10 to 15 fish were anaesthetized in 60 1 of 75 ml/1 24 MS 222 neutralized with 5N NaOH. The control solution f o r i n j e c t i n g each f i s h consisted of 0.1 cc of s t e r i l e saline and peptone (0.$5 and 0.1 percent respectively) injected i n t r a p e r i t o n e a l l y i n the lower mid-lateral body wall anter-i o r to the pel v i c f i n s . S t e r i l e , p l a s t i c 1.0 cc syringes were f i l l e d with control solution, f i t t e d with 26.5 gauge needles (both from Becton Dickinson, Rutherford, N.Y.) and kept on ice u n t i l the time of i n j e c t i o n . After i n j e c t i o n , the f i s h were placed i n a 186 1 recovery tank receiving the same water as the tes t tanks. After a group of 240 f i s h had been injected with the control solution, they were trans-ferred back into t h e i r t e s t tanks. This procedure was car-r i e d out ttiree times f o r the three control groups. The same i n j e c t i o n procedures were followed for the three experimental groups but with a suspension of k i d -ney disease bacteria (App. IIB and IIC). This inoculum was prepared with bacteria harvested (App. IIC) i n t h e i r l og phase of growth from culture media (App. IIA). The experi-mental f i s h f o r a l l toxicant l e v e l s of NaPCP received the same volume (0.1 ml) and concentration (0.1 OD, 420 nm) of inoculum as determined i n I n i t i a l Experiment A. Injections f o r both control and experimental groups were carried out on the same day. Viable counts (App. IID) indicated that approximately 41.28 x 10^ viable kidney disease b a c t e r i a l c e l l s were injected into each experimental f i s h . i TOXICANT ADMINISTRATION The time required for blood c o l l e c t i o n , measurement ?5 of haematological parameters and renewal of toxicant s o l u -tions permitted sampling from only two of the s i x tanks to be completed i n one day. Since the time course of the experimental treatments was monitored at four-day i n t e r v a l s , sampling could be accomplished f o r a l l tanks over t h i s four day period and the intersampling i n t e r v a l f o r each group of f i s h kept constant f o r the duration of the experiment. Hence the i n i t i a l administration of NaPCP to the four groups of f i s h ( F i g . I l l ) was staggered to provide equal toxicant expo-sure times to each group. It should be noted, however, that a l l the infected f i s h received b a c t e r i a l i n j e c t i o n s at the same time. Consequently, at any given sampling time the exposure to b a c t e r i a l i n f e c t i o n would be unequal by as much as three days between the groups exposed to the lowest and highest concentrations of NaPCP while toxicant exposure would be the same f o r a l l groups. Because consistent v i a b i l i t y of stored inoculum, or the v i a b i l i t y of separate cultures of bacteria grown at in t e r v a l s to f i t into the staggered sam-p l i n g schedule, could not be assured, i t was judged more p r a c t i c a l to i n j e c t the b a c t e r i a l treatment groups at the same time with one inoculum batch rather than r i s k unequal v i a b i l i t y of inocula prepared to give equal b a c t e r i a l expo-sure times. Sampling of the uninfected control and kidney disease infected experimental f i s h i n clean water commenced eight days a f t e r i n j e c t i o n . Sampling of the uninfected control and infected experimental f i s h at the intermediate (©.05 of the 96 h LC^ Q) NaPCP l e v e l commenced four days a f t e r toxicant administration and nine days a f t e r i n j e c t i o n . 26 Sampling of the uninfected control f i s h and infected experi-mental f i s h at the high (0.5 of the 96 h L C 5 0 ) NaPCP l e v e l commenced four days a f t e r toxicant administration and ten days a f t e r i n j e c t i o n . A l l control f i s h were sampled i n the morning and experimental f i s h i n the afternoon. For each experimental group, sampling was done at four-day i n t e r v a l s and a t o t a l of nine samples were completed unless prevented by mortality. The intermediate and high l e v e l s of NaPCP admini-s t r a t i o n commenced on the f i f t h and sixth days respectively a f t e r the i n j e c t i o n day. The preparation and d i l u t i o n proce-dures f o r the NaPCP are given i n Appendix IA.' The toxicant concentrations i n the t e s t tanks were allowed to equi l i b r a t e to the desired l e v e l over one replacement time (4.5 h), as done i n I n i t i a l Experiment B. SAMPLING Blood f o r the haematological measurements was coll e c t e d from the severed caudal peduncle. To provide s u f f i c i e n t blood f o r these measurements, two f i s h were r e -quired. Each sample consisted of 12 pooled haematological measurements, ie 24 f i s h . Two f i s h were netted together and k i l l e d by blows to the head. They were then blotted with paper towels and the caudal peduncle wiped with a tissue soaked with 95$ ethanol. Blood was co l l e c t e d i n 280 u l heparinized Natelson tubes (Sherwood Med. Ind. St. Louise MO) by f i l l i n g to an a r b i t r a r y mark approximately 1/4 to 1/3 the length of the tubes. A heparinized microhaematocrit tube 27 (Pre-cal, Clay Adams, N.J.) was also f i l l e d to l a t e r deter-mine the haematocrit (HCT) value. The caudal peduncle of the second f i s h was then severed and the columns of blood i n the Natelson tubes were doubled. Another microhaematocrit tube was f i l l e d f o r HCT determination as above. One of the two f i s h was put aside f o r fork length and weight measure-ments and pathological examination. The other f i s h was d i s -carded. The t o t a l time from opening of the tank cover to completion of blood withdrawal from the two f i s h took approx-imately four to s i x minutes. PARAMETER MEASUREMENTS The microhaematocrit tubes were sealed and imme-d i a t e l y centrifuged (Int. Equipt. Co., Div. of Damon) f o r 3.5 min at 13,000 rpm f o r the determination of HCT values according to Snieszko (i960). The haemoglobin (Hb) deter-mination was made from a 30 u l aliquot of whole blood trans-ferred from one of the Natelson tubes by the cyanmethaemo-globin method (Harry, 1968) using the Accustat Blood Analyzer system (Becton Dickinson, Mississauga, Ontario). Blood f o r t o t a l red and white blood c e l l counts (RBC and WBC respec-t i v e l y ) was obtained from an aliquot of whole blood from the f i r s t Natelson tube. It was collected into a standard red c e l l d i l u t i n g pipette (1:200 d i l u t i o n ) and dil u t e d with Rees-Ecker solution (Klontz and Smith, 1968). Red and t o t a l white blood c e l l counts (including thrombocytes) were made with a haemocytometer. RBC counts were multiplied by 10,000 and WBC counts by 500 to give the number of c e l l s per mnr 28; (Hesser, I960). Plasma was obtained by centrifuging the Natelson tubes (15 min at 2500 rpm) and transferred to p l a s t i c v i a l s f o r storage at -20 C. Blood urea nitrogen (BUN), glucose (GLU) and t o t a l protein (TP) determinations were made on the stored plasma samples at a l a t e r date. The determination of BUN, GLU and TP values on previously frozen plasma were determined c o l o r i m e t r i c a l l y using the Accustat Blood Analyzer System. Three erythrocytic indices were calculated from the HCT, Hb and RBC values using the following formulae: (MCHC) Mean Corpuscular Hb Concentration (pg) = Hb(g/100 ml) x 10 RBC(millions/mm 3) (MCH) Mean C e l l u l a r Hb ($) = Hb(g/100 ml) x 100 HCT ($) (MCV) Mean C e l l Volume (u 3) = HCT($) x 10 - RBC(millions/mm 3) The t o t a l time to carry out the above procedures and measure-ments took approximately 10 to 12 minutes. PATHOLOGICAL EXAMINATIONS When a sampling set had been completed, the external and i n t e r n a l pathological examinations were carr i e d out. For pathological examinations, the scales of a l l twelve f i s h from 2 9 one sample were removed from one side and the skin bathed i n 95$ ethanol. Kidney smears were a s e p t i c a l l y made by obtaining a piece of kidney tissue on a s t e r i l i z e d Nichrome inoculating loop through an i n c i s i o n below and p a r a l l e l to the l a t e r a l l i n e made with a s t e r i l i z e d s c a l p e l . The smears were Gram stained and inspected f o r the presence of kidney disease bacteria. General notes were made on external and in t e r n a l c h a r a c t e r i s t i c s of each f i s h with p a r t i c u l a r atten-t i o n f o r those pathological symptoms observed i n I n i t i a l Experiment A. The external and in t e r n a l physical character-i s t i c s were noted and were grouped a r b i t r a r i l y into s i x main categories (number coded one to s i x ; App. I l l ) according to the severity of the i n f e c t i o n . The physical examination was car r i e d out fo r each f i s h sampled and a mean was calcu-lated f o r each complete sample of twelve f i s h . This cate-gorization of pathological conditions i s tabulated i n Table I I I . DATA ANALYSIS Preliminary computation of means and analysis of variance was carr i e d out using the prepared program, ANOVAR (University of B r i t i s h Columbia). Scheffe's t e s t was used to compare differences between means fo r s t a t i s t i c a l s i g n i f i -cance at P = 0.05 (Edwards, 1967). The res u l t s of s p e c i f i c comparisons f o r sig n i f i c a n c e between means are shown on the graphs of the changes i n the haematological parameters over time (Figs. V - XII ). The following comparisons were made f o r each parameter at each sampling time: between values of 30' uninfected control f i s h and kidney disease infected experi-mental f i s h at each toxicant l e v e l ; between values of unin-fected control f i s h among the three toxicant l e v e l s and between values of kidney disease infected experimental f i s h among the three toxicant l e v e l s . The grand mean represents the mean of a l l the means f o r a l l sampling days. The mean of absolute d i f f e r -ences between control and experimental f i s h represents the mean of a l l the absolute values of the in d i v i d u a l differences between control and experimental means at each sampling time f o r a l l sampling days. These two calculated values are pre-sented i n Figures XIII to XX as a general summary of the r e l a t i v e e f f e c t s the various experimental conditions had on the measured haematological parameters f o r the experiment as a whole. In t h i s treatment of data, the value derived i s f o r the experiment as a whole rather than f o r s p e c i f i c sampling times. A greater departure of experimental f i s h value from control f i s h value i n toxicant r e l a t i v e t i clean water i s indicated by a t a l l e r bar for the mean of absolute differences between control and experimental groups f o r group 2 r e l a t i v e to group 1. 51a SECTION IV RESULTS FOR THE MAIN EXPERIMENT 31 Throughout the r e s u l t s and discussion sections, the symbols IC, 2C, 3C and IE, 2E, 3E w i l l be used to desig-nate uninfected control f i s h i n clean water, i n the i n t e r -mediate toxicant l e v e l , i n the high toxicant l e v e l , and k i d -ney disease infected experimental f i s h i n clean water, i n the intermediate toxicant l e v e l , i n the high toxicant l e v e l , respectively, as shown in the experimental design ( F i g . I I I ) . The f i s h i n a l l the tanks appeared to become well conditioned to the presence of a net i n the tanks during the 27-day acclimatization period. The f i s h were not disturbed by the presence of the sampling net; t h e i r normal behavior of rushing to the surface i n a n t i c i p a t i o n of food each time the cover was opened persisted throughout the early period of sampling. Their c h a r a c t e r i s t i c schooling of groups 2E and IE f i s h seemed to deteriorate, however, beginning on the 12th (T3) and 1 6 t h (T1+) days respectively. An i n -creasing reduction i n feeding was observed i n groups IE and 2E s t a r t i n g about the second sampling day (T2). The deter-i o r a t i o n of c h a r a c t e r i s t i c schooling was also observed i n f i s h of group 3C s t a r t i n g on the 28th day (T7) of toxicant exposure but not i n groups 2C or IC. In addition, sluggish-ness, d i s o r i e n t a t i o n and markedly reduced resistance to cap-ture became evident i n a few individuals of these groups and followed by several days, the appearance of deterioration of schooling behavior. M o r t a l i t i e s also started to occur at about these times i n these groups. It seemed l i k e l y that 32 those indiv i d u a l s exhibiting the sluggish behavior com-prised the m o r t a l i t i e s . During blood c o l l e c t i o n , c l o t t i n g frequently occurred i n a l l groups of f i s h at the commencement of the experiment. However, a f t e r the l6th day ( T 4 ) » blood c l o t -t i n g was l e s s troublesome i n both IE and 2E groups. This reduction i n c l o t t i n g tendency also occurred i n group 3C but not u n t i l approximately the 25th day of toxicant exposure. A l l the f i s h i n group 3E died suddenly on the se-cond sampling day.(T2). Although i t was noted on the pre-vious day that most of the f i s h were at the surface, feeding behavior, or t h e i r response to a routine procedure to which they had been conditioned, was normal and the sudden mor-t a l i t y of t h i s group was unexpected. Gram stained kidney smears showed that the pathogen had invaded the kidney tissu e by t h i s day (14 days a f t e r i n j e c t i o n with kidney disease b a c t e r i a ) . Therefore comparisons involving group 3E were possible f o r only the f i r s t sampling day ( T l ) . Mor-t a l i t i e s i n groups IE and 2E, due to the progression of the disease, prevented further sampling of experimental f i s h i n these groups beyond the sixth sampling day ( T 6 ) . Since there were some variations i n the nature and time courses of blood changes, they w i l l be discussed separately. 1. Comparison between uninfected control f i s h and kidney disease infected experimental f i s h f o r a l l toxicant l e v e l s In general, HCT, Hb, RBC, BUN, TP and GLU a l l showed 3 3 a general depression i n kidney disease infected f i s h r e l a -t i v e to uninfected control f i s h over the experimental period. Haematocrit Experimental f i s h i n groups IE, 2E,and 3E a l l showed decreased HCT values r e l a t i v e to sham injected con-t r o l groups IC, 2C and 3C over the incubation time of the disease. The differences were s i g n i f i c a n t from the f i r s t sampling day (Tl) for a l l groups and increased with time i n groups IC, IE and 2C, 2E ( F i g . V). Haemoglobin Experimental f i s h i n groups IE, 2E and 3E a l l showed lowered Hb values over the incubation time of the disease r e l a t i v e to sham injected control groups IC, 2C and 3C ( F i g . VI). S i g n i f i c a n t differences were observed between IC and IE groups from the f i r s t to the sixth sampling day (T l to T6). Although these differences were r e l a t i v e l y con-stant throughout the sampling days,, they increased over the same i n t e r v a l s for the 2C and 2E groups. At t h i s toxicant l e v e l , the group 2E Hb value was s i g n i f i c a n t l y greater than for group 2C f i s h on the f i r s t sampling day ( T l ) . After t h i s time, the 2E Hb values declined s i g n i f i c a n t l y below 2C values and continued to f a l l u n t i l the f i n a l sampling day (T6). On the f i r s t sampling day (Tl) the Hb value of group 3E was s i g n i f i c a n t l y lower than the control group 3C value. Red blood c e l l count Experimental f i s h i n groups IE, 2E and 3E a l l showed s i g n i f i c a n t l y lower RBC counts compared to the sham 34 injected control groups IC, 2C and 3C over the incubation time of the disease ( F i g . VII). Differences between groups 1G and IE as well as 2C and 2E were s i g n i f i c a n t on the f i r s t sampling day (Tl) and s i m i l a r l y continued to increase u n t i l the sixth sampling day. RBC values of groups 3C and 3E were s i g n i f i c a n t l y d i f f e r e n t on the f i r s t sampling day ( T l ) . Blood urea nitrogen The BUN values f o r group IE were s i g n i f i c a n t l y lower than IC values on the f i r s t two sampling days ( T l and T2; F i g . X). For the remainder of the sampling period (T3-T6), the BUN values were highly variable f o r the IE group showed an increasing trend towards the IC group values about which they fluctuated markedly from the t h i r d to the sixth sampling day (T3-T6). Two of these variations were s i g n i f i -cantly d i f f e r e n t from the group IC values on the f i f t h and sixth sampling days ( T 5 and T6) when the BUN values of the f i s h i n group IE f e l l below and then rose above the IC values respectively. The BUN values of f i s h i n group 2E, however, were s i g n i f i c a n t l y lower than those of group 3C on the f i r s t sampling day ( T l ) . Total protein Experimental f i s h i n group IE showed s i g n i f i c a n t l y lower TP values r e l a t i v e to group IC f o r the f i r s t two samp-l i n g days ( T l and T2; F i g . XI). After t h i s i n i t i a l depression TP values i n group IE f i s h were higher than group IC f i s h on the next two sampling days (T3 and T4) but then declined below the IC group values i n the l a s t two sampling days (T5 and T6). The differences between the two groups were s i g n i f i c a n t •:: ^  35 on the fourth, f i f t h and sixth sampling days (T4, T5 and T6). TP values f o r group 2 E f i s h were s i g n i f i c a n t l y lower than group 2 C f i s h f o r a l l sampling days ( T 1 - T 6 ) . The TP value of group 3E f i s h was s i g n i f i c a n t l y lower than the group 3C value on the f i r s t sampling day (TI). Glucose Experimental f i s h i n group 2 E showed GLU values that were s i g n i f i c a n t l y lower than control f i s h group 2 C f o r a l l sampling days ( T 1 - T 6 ) . The re s u l t s f o r the IE and IC groups were s i m i l a r except f o r the f i r s t sampling day (TI; Fi g . XII). The GLU value f o r f i s h i n group 3E was also s i g n i f i c a n t l y lower than the group 3C f i s h on the f i r s t sampling day (TI). Mean c e l l volume In contrast to the trend of depressed values of HCT, Hb, RBC, BUN, TP and GLU found f o r experimental f i s h during the progression of kidney disease, MCV values i n infected experimental f i s h increased above the respective uninfected control f i s h values. The MCV values i n IE group f i s h varied considerably and a s i g n i f i c a n t difference com-pared to the IC group values was not observed u n t i l the sixth sampling day (T'6) when the group IE f i s h had a higher MCV (F i g . IX). MCV values i n group 2 E f i s h were s i g n i f i -cantly higher than group 2 C f i s h values on the t h i r d , f i f t h and s i x t h sampling periods (T3, T 5 and T6). NO s i g n i f i c a n t difference was observed between f i s h i n group 3.E and 3 C on the f i r s t sampling day ( T l ) . Three blood parameters (WBC, MCHC and MCH) showed 36 v a r i a b l e r e s p o n s e s t o k i d n e y d i s e a s e b a c t e r i a l i n f e c t i o n . T o t a l w h i t e b l o o d c e l l c o u n t s E x p e r i m e n t a l f i s h i n g r o u p I E h a d s i g n i f i c a n t l y l o w e r WBC on t h e f i r s t t h r e e s a m p l i n g d a y s ( T 1-T3) r e l a t i v e t o IC g r o u p f i s h ( F i g . V I I I ) . A f t e r t h i s t i m e , t h e I E g r o u p v a l u e s c o n t i n u o u s l y i n c r e a s e d t o w a r d t h e IC g r o u p v a l u e s u n -t i l by t h e s i x t h s a m p l i n g d a y (T6) t h e I E g r o u p f i s h had a s l i g h t l y h i g h e r (P g r e a t e r t h a n .05) WBC. F o r t h e g r o u p 2E f i s h , t h e WBC was a l s o s i g n i f i c a n t l y l o w e r t h a n g r o u p 2C v a l u e s f o r t h e f i r s t t h r e e s a m p l i n g days ( T 1-T3). S i m i l a r l y , t h e WBC f o r g r o u p s 2 E f i s h i n c r e a s e d u n t i l by t h e s i x t h s a m p l i n g d a y (T6) i t was s i g n i f i c a n t l y h i g h e r t h a n t h e g r o u p 2C v a l u e . On t h e f i r s t s a m p l i n g d a y ( T l ) t h e e x p e r i m e n t a l f i s h i n g r o u p 3E showed a s i g n i f i c a n t l y l o w e r WBC r e l a t i v e t o t h e g r o u p 3C v a l u e . Mean c o r p u s c u l a r h a e m o g l o b i n c o n c e n t r a t i o n and mean c e l l u l a r  h a e m o g l o b i n Due t o t h e l a r g e v a r i a t i o n i n t h e MCHC and MCH v a l u e s o f i n f e c t e d e x p e r i m e n t a l f i s h and u n i n f e c t e d c o n t r o l f i s h o v e r t h e s a m p l i n g p e r i o d o f t h e e x p e r i m e n t , t h e s e r e -s u l t s have been p r e s e n t e d i n A p p e n d i x V . No e a s i l y d i s c e r n -a b l e p a t t e r n c o u l d be s e e n i n t h e MCHC and MCH v a l u e s f o r a n y o f t h e f i s h g r o u p s . 2 . C o m p a r i s o n among u n i n f e c t e d c o n t r o l f i s h g r o u p s f o r a l l  t o x i c a n t l e v e l s The c o m p a r i s o n f o r t h e u n i n f e c t e d c o n t r o l f i s h g r o u p s I C , 2C and 3C i n d i c a t e d t h a t s i g n i f i c a n t changes o c c u r r e d i n HCT, BUN and GLU v a l u e s i n r e s p o n s e t o t o x i c a n t '37 exposure. Haematocrit Although HCT i n f i s h of group 2C did s i g n i f i -cantly d i f f e r from group IC f i s h f o r a l l the sampling days ( T 1 - T 9 ) , the group 3C values were found to be s i g n i f i c a n t l y lower than both IC and 2C values from the f i r s t to the f i f t h sampling day (T1-T5). On the sixth sampling day (T6) the HCT value i n group 3C was s i g n i f i c a n t l y lower than the 2C ^ group value only (Fig. V). Blood urea nitrogen BUN values f o r group 2C f i s h were s i g n i f i c a n t l y higher than group IC and 3C f i s h on the f i r s t four sampling days (T1-T4; F i g . X). However no s i g n i f i c a n t difference was observed between the BUN values of IC and 3C over the same i n t e r v a l . Glucose Despite the large variations i n GLU values i n a l l control groups, there appeared to be a s i g n i f i c a n t depression in group 3C r e l a t i v e to groups IC and 2C on the f i r s t four sampling days (T1-T4; F i g . XII). On the f i r s t sampling day (TI) GLU values i n f i s h of group 3C were s i g n i f i c a n t l y lower than those of group 2C. However, on the second, t h i r d and fourth sampling days (T2, T3 and T4) the group 3C values were s i g n i f i c a n t l y lower than those of both' 2C and IC groups. Inspection of Figure XI shows that although the glucose values of uninfected control f i s h i n group IC varied l i t t l e , t h i s parameter i n groups 2C and 3C was more va r i a b l e . In group 2C f i s h however, there appeared to be a trend of 3* increasing GLU values, e s p e c i a l l y a f t e r the f i f t h sampling day (T5). 3• Comparison among kidney disease infected f i s h f o r a l l  toxicant l e v e l s , Experimental f i s h i n groups IE, 2E and 3E showed that no consistent pattern occurred i n any of the measured blood parameters over a l l the sampling days (T1-T6; Figs. V - X I I ) . The histograms (Figs. XVII, XVIII and App. V, F i g . IV) show that toxicant administration appears to have caused a greater difference between the control and experi mental f i s h values of MCV, BUN and MCH i n the intermediate l e v e l of toxicant treated groups, 2C and 2E, r e l a t i v e to the clean water groups IC and IE over the whole sampling period (T1-T6). TABLE III Physical c h a r a c t e r i s t i c s code and mortality f o r the main experiment. 4 days 8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days 0 0 0 0 0 0 0 0 c 0 b 0 0 0 0 0 0 0 0 cw TP 1.4 1.7 2.2 2.8 3.3 5.3 * * Hi 0 0 0 4 5 33 52 0 0 0 0 0 0 0 0 0 c 0 0 0 0 0 0 '3. 0 0 INT 4.8 1.8 2.3 2.9 3.1 5.9 * z> 0 0 1 2 14 48 31 0 0 0 0 0 0 0 0 0 c 0 0 3 1 0 0 11 1 8 HIGH 2.2 IT 216 * # * * X 0 CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h LC 5 Q) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = # -a = b' = ^ = 12 (two f i s h pooled f o r each measurement. ) Key for Code i n Appendix III Physical C h a r a c t e r i s t i c s M o r t a l i t y f o r that day No data due to mortality 40a Figures V to XII Graphs of the responses of measured haematological parameters i n the d i f f e r e n t groups of f i s h to the experi-mental treatments, b a c t e r i a l kidney disease i n f e c t i o n and NaPCP exposure, over the sampling period. Each point represents a mean + 1 . 9 6 standard error of the mean (n=12) . Responses i n uninfected control f i s h are desig-nated by s o l i d l i n e s and kidney disease infected f i s h by broken l i n e s , a, b and c represent clean water ( 0 x 96 h L C ^ Q ) , intermediate l e v e l of NaPCP exposure ( 0 . 0 5 x 96 h L C J Q ) and high l e v e l of NaPCP exposure ( 0 . 5 x 96 h L C ^ Q ) respectively. TI represents the f i r s t sampling day, four days'tafter beginning toxicant exposure. 2 to 9 represent subsequent sampling days. Symbols on top of each sampling day designate s t a t i s t i c a l s i gnificance ( P = 0 . 0 5 ) , by Scheffe's t e s t , between means of d i f f e r e n t groups of f i s h : * = between control and experimental f i s h , 1 = i n a, 2 = i n b, 3 = i n c; H = i n control f i s h groups, A = between a and b, B = between b and c, C = between a and c; D = i n kidney disease infected f i s h . Figure V Haematocrit Figure VI Haemoglobin Figure VII Red Blood C e l l Count Figure VIII Total White Blood C e l l Count Figure IX Mean C e l l Volume Figure X Blood Urea Nitrogen Figure XI Total Protein Figure XII Glucose 40 41 Figure VI 12 12 10 8 6 4 2 12 12 12 12 12 H B . HBC H C lBC * -4 ^ O O bfl PQ o § c 1CL + Tl 4 8 9 SAMPLING PERIOD - 4 day in t e r v a l s 42 gure VII 123 12 12 12 12 12 • - H C O o o o < W H H o O o .-3 .-1 O o o PQ Q P3 a 18CL 140 100 60 b 180_ 140 100 6 0 c 180_ 140 100L I 1 1 I I L J L Tl 2 3 7 8 $ SAMPLING PERIOD - 4 day i n t e r v a l 43/ Figure VIII *123 *12 *12 'AC a 6 . o o \ O E-i to O o 1-3 1-1 w o o o o 1-1 m w E-" H H i o c 7 SAMPLING PERIOD - k- day int e r v a l s 4 4 gure IX *2 *12 HAC HB a 360^ OA I t-1 O > FI I-1 w o c 300_ 240 180L L I 1 » 1 I I _ l I Tl 2 3 4 5 6 7 8 9 SAMPLING PERIOD - 4 day int e r v a l s 4 5 * * * * * * 123 12 2 2 12-12 A B A B A B " A B C " A C n B C SAMPLING PERIOD - 4 day in t e r v a l s 46 Figure XI *123 *12 *2 *12 *12 *12 HABC HBC HBC HAB HBC DBC DA DA I I 1 L_ Tl 2 3 4 5 6 7 8 SAMPLING PERIOD - 4 day int e r v a l s 47 Figure XII *23 *12 *12 *12 *12 *12 HAB HBC HBC HBC AB 'ABC S o o S W CO O O !=> O a 75 65 55 45 35 25l b 110 105 95 85 75 65 55 45 35 25 SAMPLING PERIOD - 4 day int e r v a l s 1 48a Figures XIII to XX Histograms showing grand means and means of absolute differences between control and experi-mental f i s h f o r the d i f f e r e n t groups of f i s h . Sample sizes used to derive these values are i n -dicated on top of bars. Figure XIII Figure XIV Figure XV Figure XVI Figure XVII Figure XVIII Figure XIX Figure XX Haematocrit Haemoglobin Red Blood C e l l Count Total White Blood C e l l Count Mean C e l l Volume Blood Urea Nitrogen Total Protein Glucose HAEMATOCRIT -grand means ($) • i " I I ' i i i ' o ^ 0 0 £ ; * means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and e x p e r i m e n t a l f i s h . HAEMOGLOBIN -grand means (g/100 ml) * means o f absolute d i f f e r e n c e s between c o n t r o l and experimental f i s h . * means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and e x p e r i m e n t a l f i s h . CD WHITE BLOOD. CELL COUNT -grand means (cells/mnr/500) o ho •1 1 1 1 Co co CD Co >0 o * means of absolute differences between control and experimental f i s h . * means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and. e x p e r i m e n t a l f i s h . BLOOD UREA NITROGEN -grand means (mg/100 ml) * means of absolute differences between control and experimental f i s h . TOTAL PROTEIN -grand means (gm/lOO ml) CD h-f rO * o CO col * N5 * means of absplute differences between control and experimental f i s h . * means o f a b s o l u t e d i f f e r -e n c e s between c o n t r o l and e x p e r i m e n t a l f i s h . 56 Table IV Comparison of various haematological values mea-sured i n t h i s study with those of Thomas et. a l . (1969). This study Thomas et. a l . (grand means of (1969)(means; uninfected control male - female) f i s h i n clean water) HCT ($) 34.64 41.0 - U.6 Hb (g/100 ml) 3 , 2 6 7.9 8.2 RBC (cells/mm3/lO,000) 150.93 157.40 - 156.10 MCV (u 3) 237.08 261 - 268 MCHC (pg) 55.92 "50.1 - 52.8 MCH ($) 23.96 19.2 - 19.7 TP (g/100 ml) 3.78 4.02 - 4.12 GLU (mg/100 ml) 56.69 100.8 - 99.8 57a SECTION V DISCUSSION 57 The r e s u l t s of the main experiment indicate that the experimental factors of NaPCP exposure and kidney d i -sease i n f e c t i o n imposed s t r e s s f u l conditions on the f i s h and that these factors acted s y n e r g i s t i c a l l y . The occur-rence of m o r t a l i t i e s , as shown i n Table I I I , supports t h i s contention. The fact that the mo r t a l i t i e s for groups IE and 2E started to occur at approximately the same time suggests that the intermediate toxicant l e v e l exposure did not enhance the progression of the kidney disease to a point where a difference i n commencement or rate of mor-t a l i t y occurred. However, the catastrophic mortality that occurred i n group 3E f i s h indicates that the high l e v e l of NaPCP imposed a stress on the f i s h i n t h i s group, i n addi-t i o n to the i n f e c t i o n , that was beyond t h e i r adaptive a b i l i t i e s , thus culminating i n death. Earp et_. a l . (1955) reported several catastrophic m o r t a l i t i e s i n adult chinook salmon due to t h i s disease. In one of these instances, the uncontrolled sudden out-break occurred a f t e r these f i s h were transported and tem-pered into sea water. The transportation and handling may have caused a stress that played a role s i m i l a r to that of the high l e v e l of toxicant exposure i n t h i s experiment i n contributing to the catastrophic mortality. The fact that m o r t a l i t i e s occurred and increased i n f i s h i n groups 2C and 3C indica'tes that the two concentrations of NaPCP i n t h i s study became s t r e s s f u l to the f i s h with continuous exposure over time. The e a r l i e r commencement and greater magnitude 5* o f t h e m o r t a l i t i e s i n g r o u p 3C f i s h compared t o g r o u p 2C f i s h i n d i c a t e t h a t t h e s t r e s s imposed by NaPCP was p r o p o r -t i o n a l t o t h e c o n c e n t r a t i o n . The p h y s i c a l c h a r a c t e r i s t i c s o f t h e i n f e c t i o n , as shown i n T a b l e I I I , i n d i c a t e t h a t d i s e a s e p r o g r e s s i o n was s l i g h t l y more a d v a n c e d i n t o x i c a n t e x p o s e d e x p e r i m e n t a l f i s h compared t o e x p e r i m e n t a l f i s h i n c l e a n w a t e r . T h i s s u g g e s t s t h a t t h e p r o g r e s s i o n o f k i d n e y d i s e a s e was e n -h a n c e d w i t h NaPCP e x p o s u r e and t h a t t h i s e f f e c t a p p e a r s t o have been p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f N a P C P . The s t r e s s o f NaPCP e x p o s u r e was t h o u g h t t o have r e d u c e d t h e r e s i s t a n c e o f t h e i n f e c t e d e x p e r i m e n t a l f i s h i n g r o u p s 2E and 3E t o t h e d e b i l i t a t i v e e f f e c t s o f t h e p a t h o g e n , t h u s e n h a n c i n g t h e p r o g r e s s i o n o f t h e d i s e a s e . A l t h o u g h t h e e x p e r i m e n t a l t r e a t m e n t s a p p e a r t o have imposed v a r y i n g d e g r e e s o f s t r e s s on f i s h i n g r o u p s I E , 2C, 2E, 3C and 3E, t h e a m b i e n t c o n d i t i o n s o f t h e e x p e r i -ment were c o n s i d e r e d n o t t o have s i g n i f i c a n t l y s t r e s s e d t h e f i s h i n a n y o f t h e g r o u p s . T h i s was d e t e r m i n e d o n t h e b a s i s t h a t t h e r e i s a g e n e r a l agreement between t h e h a e m a t o l o g i c a l p a r a m e t e r v a l u e s o b t a i n e d i n t h i s s t u d y and t h a t o f Thomas ( 1 9 6 9 ; T a b l e l y ) f o r f i s h o f t h e same s p e c i e s and a g e . S l i g h t d i s c r e p a n c i e s between t h e v a l u e s o f t h e two s t u d i e s were a t t r i b u t e d t o t h e p o s s i b l e d i f f e r e n c e i n u n c o n t r o l l e d a m b i e n t c o n d i t i o n s a s w e l l as d i f f e r e n c e s i n measurement t e c h n i q u e s . The r e l a t i v e l y h i g h f l o w - t h r o u g h r a t e s i n t h e t e s t t a n k s and t h e a c c l i m a t i z a t i o n p r o c e d u r e s were c o n s i d e r e d t o have a m e l i o r a t e d f a c t o r s t h a t o t h e r w i s e may have imposed 59' a d d i t i o n a l s t r e s s f u l conditions f o r the f i s h . As indicated i n Table I I I , the GLU values obtained i n t h i s study were lower than the value obtained by Thomas (1969) f o r t h i s species. This difference was also a t t r i -buted to some persistent difference i n ambient conditions i n the two studies. As w i l l be discussed i n t h i s section, GLU l e v e l s have been found to be very sens i t i v e indicators of s t r e s s f u l conditions and that hyperglycemia i s usually a metabolic response to stress i n f i s h . BUN and WBC count values have not been previously reported i n the l i t e r a t u r e f o r t h i s species at t h i s age. Eight of the ten haematological parameters that were measured i n the main experiment showed consistent pat-terns over the sampling period i n response to the experi-mental treatments. These sublethal blood changes w i l l be discussed according to the nature of t h e i r responses. Haemodilution was thought to be the primary cause f o r the generally lower HCT, Hb, RBC, BUN, TP and GLU values f o r kidney disease infected experimental f i s h compared to sham injected control f i s h over the sampling period (Figs. V, VI, VII, X, XI and XII). Wood and Yasutake (1955) stated that they thought the tissue of the anterior head kidney, the major haematopoietic tissue i n f i s h , was quite l i k e l y the f i r s t t i s s u e affected i n kidney disease i n f e c t i o n . The haema-to p o i e t i c tissue was always i n an advanced stage of destruc-t i o n even i n the e a r l i e s t stages of the disease. They ob-served extensive f i b r o t i c lesions enveloping renal tissue i n t h i s part of the kidney. 60 Furthermore, as the disease progressed, they observed destruction of the posterior portions of the kidney which took the form of a more general tissue reaction. The posterior kidney i s primarily involved i n regulating body f l u i d s and el e c t r o l y t e s as well as being an excretory organ (Hickman and Trump, 1969). Wood and Yasutake (1955) also observed severe destruction of splenic t i s s u e , another haematopoietic tissue i n f i s h , as a consequence of kidney disease i n f e c t i o n . The symptoms of kidney disease observed i n I n i -t i a l Experiment A and the main experiment i n t h i s study are in agreement with the observations of Wood and Yasutake (1955). The physical c h a r a c t e r i s t i c s , as presented i n Table I I I , of these experimental f i s h on the f i r s t sampling day (Tl) indicates that some degree of breakdown i n body f l u i d regulation was present at t h i s time. With the degeneration of excretory t i s s u e , f i s h i n freshwater would tend to imbibe more water from t h e i r hypotonic environment than they could excrete, thus r e s u l t i n g i n a haemodilution. The i n h i b i t i o n of erythropoiesis by the destruction of haematopoietic tissue would contribute to t h i s d i l u t i o n effect with regards to HCT, Hb and RBC count values i n i n -fected experimental f i s h r e l a t i v e to uninfected control f i s h . Additional factors such as leakage through open lesions and possible reduced l i v e r function were considered to have contributed to the s i g n i f i c a n t l y lower TP values i n kidney disease infected experimental f i s h compared to uninfected control f i s h over time. Hunn (1964) observed depressed TP 61 values i n kidney diseased f i s h r e l a t i v e to uninfected con-t r o l f i s h . He also attributed t h i s effect to the factors mentioned here. Reduced TP values i n f i s h stressed by other pathogens have been reported by several workers (Shieh and Maclean, 1 9 7 6 ; Mulcahy, 1 9 6 7 , 1 9 7 1 ; Yamashita, 1 9 6 7 ; Einszporen-Orecka, 1 9 7 0 ) . There were two a d d i t i o n a l factors that were con-sidered to have contributed to the s i g n i f i c a n t l y depressed GLU values i n infected experimental f i s h compared to unin-fected control f i s h . These were the possible depletion of glycogen stores as a r e s u l t of the demand the multiplying pathogens imposed on t h e i r hosts' energy resources and the fact that cessation of feeding occurred i n f i s h i n groups IE and 2E from about the second sampling day. MCV values i n kidney disease infected experi-mental f i s h were generally higher than uninfected control f i s h over the sampling period ( F i g . IX). This s i g n i f i c a n t increase was considered to be the r e s u l t of the red blood c e l l s imbibing water from the surrounding plasma that was becoming increasingly hypotonic to the c e l l s due to the haemo-d i l u t i o n already discussed. I f the rate of decrease i n HCT ' values was l e s s than the rate of decrease i n RBC counts, an increase i n the calculated MCV values, f o r the experimental f i s h r e l a t i v e to control f i s h , would r e s u l t . Erythrocytic swelling i s one way i n which t h i s d i f f e r e n t i a l decrease can occur. Furthermore, i n h i b i t i o n of erythropoiesis by haema-to p o i e t i c t i s s u e damage would tend to cause larger, more mature erythrocytes to predominate i n the peripheral 62 blood picture. This l a t t e r f actor may also have contributed to the increased MCV values i n kidney disease infected f i s h compared to uninfected control f i s h . The variable response seen i n WBC counts to kidney disease i n f e c t i o n over the sampling period ( F i g . VIII) was a t t r i b u t e d to a possible stress-mediated leucopenia coupled with haemodilution, i n the i n i t i a l sampling days, followed by a possible increase i n c i r c u l a t i n g neutrophiles i n response to the increasing t i s s u e damage caused by the pathogen. I t was thought that i n the i n i t i a l sampling days (T1-T3), the pathogen or one or more of the physiological e f f e c t s of the i n f e c t i o n e l i c i t e d a non-specific stress, response i n experi-mental f i s h . The phenomenon of WBC count depression i n response to s t r e s s f u l s ituations and to adrenocorticotrophic hormone (ACTH) or c o r t i c o s t e r o i d administration i n other t e l e o s t s have been reported by several workers (Weinreb, 1958; McLeay, 1973 a, b, 1975 a, b; Benet and N e v i l l e , 1975). McLeay (1973 a), using the technique of d i f f e r e n t i a l c e l l counting, determined the primary cause of leucopenia i n coho salmon stressed with a 12 h exposure to one h a l f the 96 h LC^ Q of k r a f t pulp m i l l effluent to a reduction i n the number of c i r c u l a t i n g small lymphocytes. He suggested t h i s lympho-penic response was due to a stress-mediated increase i n c o r t i c o s t e r i o d secretion by the i n t e r r e n a l t i s s u e . Both stress and c o r t i c o s t e r i o d administration have been shown to cause lymphopenia i n salmonids (Weinreb, 1958. McLeay, 1970, 1973 a, b) and i n other vertebrates (Dougherty, 63 I960; Benett and Harbottle, 1968; Benett et. a l . , 1972) where the lymphocytes have been shown to be susceptible to l y s i s by c o r t i c o s t e r o i d s . The ascending WBC counts a f t e r the t h i r d sampling day (T3) i n infected experimental f i s h were thought to be due to an increase i n c i r c u l a t i n g neutrophiles i n response to the increasing tissue damage caused by the p r o l i f e r a t i o n of the kidney disease bacteria throughout the f i s h ' s body with time. McLeay (1973 a) stated that "In both mammals and tele o s t f i s h , the number of c i r c u l a t i n g neutrophiles are apparently unaffected by c o r t i c o s t e r o i d administration a l -though elevated by both stress and ACTH injections (Dougherty and White, 1943, 1944; Weinreb, 195#); the neutrophile, there-fore i s not thought to be under dire c t adrenocortical con-t r o l . " It i s known i n mammalian physiology that a marked increase i n c i r c u l a t i n g neutrophiles occurs i n response to tissue damage. In mammals, a globulin substance known as leucocytosis promoting factor i s produced and released by most damaged or inflamed tissues and causes a release of "reserved" neutrophiles from the bone marrow which may har-bour up to 30 or 40 times the number of c i r c u l a t i n g neutro-philes as well as an increase i n the production of t h i s type of repair c e l l . Although the responses of the blood parameters observed i n t h i s study are i n agreement with those reported by other workers, several contrary observations were made i n t h i s study. For example, depressed BUN values f o r kidney 6 4 ' -d i s e a s e i n f e c t e d f i s h compared t o u n i n f e c t e d c o n t r o l f i s h ( F i g . X ) a r e c o n t r a r y t o t h e o b s e r v a t i o n s o f s e v e r a l r e p o r t s where e l e v a t e d BUN v a l u e s have been o b s e r v e d i n f i s h e x h i b i t i n g p a t h o l o g i c a l c h a r a c t e r i s t i c s due t o o t h e r d i s e a s e s ( S h i e h , 1 9 7 6 ; Y a m a s h i t a , 1 9 6 7 ; F i e l d e t . a l . , 1 9 4 4 ) . However t h e known h i s t o p a t h o l o g i c a l e f f e c t s o f t h i s s p e c i f i c p a t h o g e n , as d i s c u s s e d a b o v e , s u p p o r t t h e e x p l a n a t i o n p r e s e n t e d h e r e as a p o s s i b l e mechanism o f t h e o b s e r v e d r e s u l t . The e l e -v a t e d BUN l e v e l s i n g r o u p 2C f i s h r e l a t i v e t o g r o u p 1G and 3C f i s h may have been a s p e c i f i c p h y s i o l o g i c a l r e s p o n s e t o t h e NaPCP o r t o a g e n e r a l s t r e s s r e s p o n s e . Wedemeyer ( 1 9 7 0 ) f o u n d t h a t a u r e m i a d e v e l o p e d i n r a i n b o w t r o u t e x p o s e d t o u n n e u t r a l i z e d MS 222. I t was u n c l e a r i n ' h i s s t u d y w h e t h e r t h i s t r e n d was due t o a c c e l e r a t e d p r o d u c t i o n o r r e t a r d e d e x c r e t i o n . He c o n s i d e r e d d e c r e a s e d g i l l t r a n s p o r t due t o t h e MS 222 ( s u l f o n i c a c i d ) u n l i k e l y s i n c e p l a s m a c h l o r i d e l e v e l s were u n a f f e c t e d . W i t h t h e v e r y l i m i t e d k n o w l e d g e o f t h e e f f e c t s o f t h e t o x i c a n t , NaPCP, o n t h e p h y s i o l o g y o f f i s h , i t i s d i f f -i c u l t t o s p e c u l a t e on t h e p o s s i b l e mechanisms r e s p o n s i b l e f o r t h e e l e v a t e d BUN l e v e l s . The f a c t t h a t an e l e v a t i o n o f t h i s p a r a m e t e r o c c u r r e d i n i n f e c t e d e x p e r i m e n t a l f i s h i n g r o u p I E s u g g e s t s t h a t t h i s e l e v a t i o n may n o t have been due t o t h e s i n g u l a r e f f e c t o f t h e t o x i c a n t . The p e c u l i a r f a c t t h a t t h e l e v e l s f o r f i s h i n g r o u p 3C d i d n o t s i g n i f i c a n t l y d e v i a t e f r o m t h e f i s h i n g r o u p I C , w i t h t h e e x c e p t i o n o f t h e f o u r t h , f i f t h and s e v e n t h s a m p l i n g d a y s (T4, T5 and T7) w h i c h p r o b a b l y were a c o n s e q u e n c e o f t h e s e v e r e f l u c t u a t i o n s , 65 i s not explained. The intermediate l e v e l of toxicant exposure e l i c i t e d a s l i g h t hyperglycemic response i n group 2C f i s h r e l a t i v e to group IC f i s h a f t e r the f i f t h sampling day (T5) ( F i g . XII). This i s i n agreement with several workers who have reported hyperglycemia i n several species of f i s h i n response to various s t r e s s f u l factors such as transport, muscular exercise, handling, capture and anaesthesia (Scott, 1921; Simpson, 1926; Menten, ,1927; Chavin and Young, 1970; Black, 1957 a, b, c; Black et. a l . , I960; Wedemeyer, 1972; Wardle, 1972 and Houston et. a l . , 1971 a, b; Soivio and O i k a r i , 1976). Hyperglycemia has also been reported as response i n f i s h to toxicant exposure (Hunn, 1972; McLeay, 1973, 1974, 1975). McLeay (1975) interpreted t h i s hyperglycemia as being the r e s u l t of glucocorticoid and catecholamine secretion from the i n t e r r e n a l tissue i n response to s t r e s s f u l s t i m u l i . The s l i g h t hyperglycemic response i n group 2C f i s h i s also i n t e r -preted as being due to a s t r e s s f u l condition developing with time and the possible increase i n the secretion of these "stress hormones" from the int e r r e n a l t i s s u e . Contrary to these observations, the GLU value i n group 3C f i s h was s i g n i f i c a n t l y lower than the GLU value i n group IC f i s h on the f i r s t sampling day ( T l ) . This observa-t i o n was interpreted as being due to the oxidative uncoupling action of the NaPCP (Alderdice, 1963) increasing the metabolic rate of the f i s h and thereby increasing the uptake of GLU from the peripheral c i r c u l a t o r y system. 66 As seen i n the m o r t a l i t i e s and physical charac-t e r i s t i c s (Table I I I ) , a synergistic effect was also ob-served between the experimental factors of kidney disease i n f e c t i o n and NaPCP exposure i n the sublethal responses of several of the haematological parameters over time. The accentuated difference i n MCV and BUN values between groups 2C and 2E f i s h compared to groups IC and IE (Figs. XVII and XVIII) f i s h indicate that the toxicant admini-s t r a t i o n enhanced the progression and subsequent effects of kidney disease i n f e c t i o n . This was interpreted as possibly being due to the stress of NaPCP exposure reducing the r e s i s -tance of the infected experimental f i s h to the d e b i l i t a t i v e e f f e c t s of the pathogen. On the basis of the physical charac-t e r i s t i c s presented i n Table I I I , i t was thought that renal damage due to the i n f e c t i o n was s l i g h t l y more advanced i n group 2E f i s h than group IE f i s h . I f t h i s were the case, the r e s u l t i n g haemodilution would be greater i n group 2E f i s h compared to group IE f i s h r e s u l t i n g i n a greater hypotonicity i n the blood of the f i s h i n the former group. This increased hypotonicity could cause the greater MCV. The fact that the ascending trend i n WBC counts of group 2E f i s h started e a r l i e r i n time and continued at a greater rate compared to group IE f i s h also suggests that toxicant administration enhanced the ef f e c t s of the disease process. The possible mechanism by which t h i s could occur has been discussed. 67a S E C T I O N V I G E N E R A L C O N C L U S I O N S A N D R E C O M M E N D A T I O N S -67 The following general conclusions were made based on the r e s u l t s of t h i s study. The two noxious agents employed i n t h i s study were determined to be s t r e s s f u l based on m o r t a l i t i e s i n response to both treatments. In response to the stress of i n f e c t i o n , consistent and s i g n i f i c a n t trends occurred i n eight (HCT, Hb, RBC, BUN, TP, GLU, MCV and WBC) out of the ten blood para-meters measured i n experimental f i s h r e l a t i v e to.sham i n -jected control f i s h . In response to the toxicant exposure, three (HCT, BUN and GLU) out of the ten blood parameters of uninfected control f i s h showed s i g n i f i c a n t trends. There-fore, i t was concluded that certain blood parameters of juvenile chinook salmon yearlings drh exhibit a response to the stress imposed by the b a c t e r i a l pathogen and the environ-mental toxicant used i n t h i s study. The fact that most of the differences between the blood parameter values.~of experimental and control f i s h were s i g n i f i c a n t on the f i r s t sampling day when no overt symptoms of the disease or the toxicant were evident suggests that these parameters have a p o t e n t i a l i n being used as early indicators of s t r e s s f u l states i n t h i s species. Several of the blood parameters showed accentuated differences between experimental and control values with toxicant administration (BUN and MCV) and s i g n i f i c a n t d i f f -erences among uninfected control values (HCT, BUN and GLU) with the three l e v e l s of toxicant administration. The phy-s i c a l c h a r a c t e r i s t i c s also indicate that the state of 68 physical d e b i l i t a t i o n caused by the pathogen was more ad-vanced i n the toxicant administered experimental f i s h com-pared to the experimental f i s h i n clean water. Further-more, although the m o r t a l i t i e s i n the experimental f i s h i n clean water and the intermediate toxicant l e v e l started to occur at approximately the same time, the catastrophic mor-t a l i t y i n the experimental f i s h at the high toxicant l e v e l was attr i b u t e d to the synergistic effects of the kidney disease b a c t e r i a l i n f e c t i o n and NaPCP exposure. These con-siderations led to the main conclusion that the environ-mental stress reduced the resistance of the f i s h to the effe c t s of the pathogen. Several recommendations can be made with regard to estimating the physiological condition of f i s h stocks (experimental, cultured or w i l d ) . The following c r i t e r i a were used i n sele c t i n g the best parameters f o r routine moni' t o r i n g of the health status i n t h i s species: - s e n s i t i v i t y to s t r e s s f u l environmental conditions - low v a r i a b i l i t y - measurement techniques which require r e l a t i v e l y i n -expensive materials that are r e a d i l y available com-mercially - t e c h n i c a l l y simple to measure Haematocrit, red blood c e l l count, mean c e l l volume and t o t a l / d i f f e r e n t i a l white blood c e l l count determinations were considered to be the best parameters to monitor. Rou-tine measurements of these parameters once a week during the summer and winter months and twice to three times a 6 9 week during the f a l l and spr ing months, when environmental condit ions are more v a r i a b l e , would enable the ear ly detec-t i o n o f s t r e s s fu l s tates , that may predispose the f i s h to disease, and the ear ly development of remedial programmes to combat -or remove the s tress . . The re su l t s o f the present study indicate that the p h y s i o l o g i c a l condi t ion of f i s h i s an important fac tor to consider i n t o x i c i t y studies such as bioassays. For the purposes of making meaningful comparisons-between d i f f e rent bioassay r e s u l t s , care must be exercised i n consider ing the various factors that contribute to the v a r i a b i l i t y i n the r e s u l t s . Some of these factors are : f i s h loading dens i ty , f i s h s ize and age, photoperiod, f i s h handl ing , exposure to v i s u a l s t re s s , temperature, acc l ima-t i o n , water hardness, pH and aerat ion (Davis and Hoos, 1 9 7 5 ) . Evidence i s presented i n t h i s study that indicates that the heal th status of the te s t f i s h may also be an important fac tor i n t h i s regard. Inspecting gram sta ins of kidney smears o f te s t f i s h as a routine procedure i n bioassays may a id i n the detect ion of abnormal stocks of f i s h and may a id the meaningful-comparisons o f d i f f e rent test r e su l t s i n the future . 70a SECTION v n BIBLIOGRAPHY 70" Akitake, H. and K. Kobayashi. 1975. Studies on the meta-bolism of chlorophenols i n f i s h - I I I . I s o l a -t i o n and i d e n t i f i c a t i o n of a conjugated PGP ex-creted by g o l d f i s h . B u l l . Jap. Soc. S c i . F i s h . , 41(3): 321-327. Alderdice, D.F. 1963. Some effects of simultaneous v a r i -ation i n s a l i n i t y , temperature and dissolved oxy-gen on the resistance of juvenile coho salmon (Oncorhynchus kisutch) to a toxic substance. Ph.d. t h e s i s , University of Toronto, 177 p. American Public Health Assoc. 1971. "Standard Methods for the Examination of Water and Wastewater", 13th Ed. Joint Publication of American Public Health Assoc., American Waterworks Assoc. and the Water P o l l u t i o n Control Federation, Washington, D.C., 874 p. Bang, F.B. 1970. Disease mechanisms i n crustaceans and marine arthropods. Pages 3#3-404 in_ Stanislas F. Snieszko, ed. A symposium on diseases of fishes and s h e l l f i s h e s . Am. Fi s h . Soc. Spec. Publ. No. 5. B e l l , G.R. 196l. Two epidemics of apparent kidney disease i n cultured Pink salmon (Oncorhynchus gorbuscha). J. F i s h . Res. Board Can. 18(4): 559-562. Bendele, R.A. and G.W. Klontz. 1975. Histopathology of teleo s t kidney diseases. Pages 365-383 i n W.E. Rubelin and G. Migaki eds. The Pathology of Fishes. The University of Wisconsin Press, Madison. Benett, M.F. and Harbottle. 1968. The effect of hydrocortisone on the blood of tadpoles and frogs, Rana castesbeiana. B i o l . B u l l . 135: 92-95. Benett, M.F., C.A. Gaudio, A.O. Johnson, and J.H. Spisso. 1972. Changes i n the blood of newts, Notophthalmus v i r i -descens, following the administration of hydro-cortisone. J . Comp. Physiol. #0: 233-237. Benett, M.F. and C.G. N e v i l l e . 1975. E f f e c t s of cold shock on the d i s t r i b u t i o n of leucocytes i n go l d f i s h , Carassius auratus. J . Comp. Physiol. 98: 213-216. 71 Black, E.C. 1957 a. Alterations i n the blood l e v e l of l a c t i c acid i n certa i n salmonoid fishes following mus-cular a c t i v i t y . I. Kamloops trout, Salmo ga i r d n e r i . J . F i s h . Res. Board Can. 14: 117-134. 1957 b. Alterations i n the blood l e v e l of l a c t i c acid i n certa i n salmonoid fishes following mus-cular a c t i v i t y . I I . Lake trou t , Salvelinus namay-cush. J . Fi s h . Res. Board Can. 14: 645-649. 1957 c. Alterations i n the blood l e v e l of l a c t i c - acid i n certa i n salmonoid fishes following mus-cular a c t i v i t y . I I I . Sockeye salmon, Oncorhynchus  nerka. J . Fi s h . Res. Board Can. 14: 807-314. Black, E.C., A.C. Robertson, A.R. Hanslip and W.G. Chiu. I960. Alterations i n glycogen, glucose and lac t a t e i n rainbow and Kamloops trout, Salmo gairdneri, f o l -lowing muscular a c t i v i t y . J . F i s h . Res. Board Can. 17: 487-500. Bullock, G.L., H.M. Stuckey and K. Wolf. 1975. Ba c t e r i a l kidney disease of salmonid f i s h e s . U.S. Dept. Int. Fis h . Dept. Leaflet : 41 7 p. Chavin, W. and J.E. Young. 1970. Factors i n the determina-t i o n of normal serum glucose l e v e l s of go l d f i s h , Carassius auratus L. Comp. Biochem. Physiol. 33: 629-653. Dougherty, T.F. and A. White. 1943. Influence of adrenal c o r t i c a l secretions on blood elements. Science,98: 367-369. . 1944. Influence of hormones on lymphoid tissue structure and function. The role of the p i t u i t a r y adrenotrophic hormone i n the regulation of the lymphocytes and other c e l l -u l a r elements of the blood. Endocrinology, 35: 1-14. Dougherty, T.F. I960. Lymphocytokaryorrhetic e f f e c t s of adrenocortical s t e r o i d s . Pages 112-124 i n J.W. Rebuck, ed. The lymphocyte and lymphocytic t i s s u e . Harper (Hoeber), New York, N.Y. 72 Davis. J.C. and R.A.W. Hoos. 1975. Use of sodium penta-chlorophenate and dehydroabietic acid as refer-ence toxicants for salmonid bioassays. J. Fish. Res. Board Can. 32: 411-416. Earp, B.J., C.H. E l l i s and E.J. Ordal. 1955. Kidney disease in young salmon. Wash. Dept. Fish. Spec. Rep. Ser. No. 1, 74 p. Einszdorn-Orecka, T. 1970. Quantitative changes in the c i r -culating blood of tenches (Tinea tinea L.)-in-fested by Ergasilus sieboldi Nordm. Pol. Arch. Hydrobiol. 17(30): 463-481. Evelyn, T.P.T., G.E. Hoskins, and G.R. Bell. 1973. First record of bacterial kidney disease in an apparently wild salmonid in British Columbia. J. Fish. Res. Board Can. 30(10): 1578-80. Field, J.B., LL. Gee, C.A. Evehjem and C. Juday. 1944. The blood picture in frunculosis induced by Bacterium  salmonicida in f i s h . Arch. Biochem. 3: 277-284. Henry, T. „ 1968. C l i n i c a l Chemistry - Principles and Technics. Harper and Row, N.Y., 742 p. Hesser, E.F. I960. Methods for routine fish haematology. Prog. Fish Cult. 22: 164-171. Hickman, CP. and B.F. Trump. 1969. The Kidney. Pages 9-239 in W.S. Hoar and D.J. Randall, ed. Fish Physiology. Vol. 1. Academic Press, Inc., New York. H i l l , H.W. 1908. The mathematics of bacterial count. Anu-: Publ. Health Ass. Rep. Bost. 33(2): 110-120. Hoben, H.J., S.A. Ching, L.J. Casarett, R.A. Young. 1976. Study of pentachlorophenol by rats. I. Method for determination of pentachlorophenol in rat plasma urine and tissue and in aerosol samples. Bull. Environ. Contamin. Toxicol. 15(1): 78-36. Houston, A.H., M.A. DeWilde and J.A. Madden. 1971 a. Some physiological effects of handling and tricane methanesulphonate anaesthetization upon the brook trout. J. Fish. Res. Board Can. 28: 625-633. V 73 Houston, A.H., J.A. Madden, R.J. Woods and H.M. Miles. 1971 b. Variations in the blood and tissue chemistry of brook trout, Salvelinus fontinalis, subsequent to handling, anaesthesia and surgery. J. Fish. Res. Board Can. 28: 635-642. Hunn, J.B. 1972. Blood chemistry values for some fishes of the upper Mississippi River. J. Minn. Acad. Sci. 38: 19-21. 1964. Some patho-physiologic effects of bacterial kidney disease in brook trout. Proc. Soc. Exp. Biol. Med. 117: 383-3^5. Iwama, G.K., G.L. Greer and P.A. Larkin. 1976. Changes in some haematological characteristics of coho salmon (Oncorhynchus kisutch) in response to active expo-sure to dehydroabietic acid (DHAA) at different exer-cise levels. J. Fish. Res. Board Can. 33(2): 285-289. Klontz, G.W. and L.S. Smith. 1968. Methods of using fis h as biological research subjects. Pages 325-385 in W.I. Gay, ed. Methods of animal experimentation. Vol III. Academic Press. Inc., New York, N.Y. Kobayashi, K. and H. Akitake. 1975 a. Studies on the meta-bolism of chlorophenols in fish - I. Absorption and excretion of PCP by goldfish. Bull. Jap. Soc. Sci. Fish. 4KD: 87-92. . 1975 b. Studies on the meta-bolism of chlorophenols in fish - II. Turnover of absorbed PCP in goldfish. Ibid. 41(1): 93-99. . 1975 c. Studies on the meta-bolism of chlorophenols in fish - IV. Absorption and excretion of phenol by'goldfish. Ibid. 41(12): 1271-1276. Kobayashi, K., H. Akitake, C. Matsuda and S. Kimura. 1975. Studies on the metabolism of chlorophenols in fish -V. Isolation and identification of a conjugated phenol excreted by goldfish. Ibid. 41(12): 1277-1282. 74 V Kobayashi, K., H. Akitake and S. Kimura. 1975. Studies on the metabolism of chlorophenols in fish - VI. Turnover of absorbed phenol in goldfish. Ibid. 42(1): 45-50. Kobayashi, K., S. Kimura and H. Akitake. 1976. Studies on the metabolism of chlorophenols in fish - VII. Sulfate conjugation of phenol and PCP in fish l i v e r s . Ibid. 42(2): 171-177. McLeay, D.J. 1970. A histometric investigation of the ac-t i v i t y of the pituitary - interrenal axis in juvenile coho salmon, Oncorhynchus kisutch Walbaum. Ph.d. Thesis. University of British Columbia, Vancouver, B.C. 189 p. . 1973 a. Effects of a 12-hour and 25-day expo-sure to kraft pulp mill effluent on the blood and tissues of juvenile coho salmon (Oncorhynchus  kisutch). J. Fish. Res. Board Can. 30: 395-400. . 1973 b. Effects of ACTH on the pituitary -interrenal axis and abundance of white blood c e l l types in juvenile coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 21: 431-440. . 1975 a. Sensitivity of blood c e l l counts in juvenile coho salmon (Oncorhynchus kisutch) to stressors including sublethal concentrations of pulp mill effluent and zinc. J. Fish. Res. Board Can. 32: 2357-2364. . 1975 b. Variations in the pituitary - inter-renal axis Nand the abundance of circulating blood-c e l l types in juvenile coho salmon, Oncorhynchus  kisutch, during stream migration. Can. J. Zool. 53: 1832-1891. Menten, M.L. 1927. Changes in:,the blood sugar of the cod, sculpin and pollack during asphyxia. J. Biol. Chem. 72: 249-253. Mulcahy, M.F. 1967. Serum protein changes in diseased Atlantic salmon. Nature 215: 143-144. \ 75 Mulcahy, M.F. 1971. Serum protein changes associated with ulcerative dermal necrosis (UDN) in the trout Salmo trotta L. J. Fish Biol. 3: 199-201. Rucker, R.R., A.F. Bernier, W.J. Whipple, and R.E. Burrows. 1951. Sulfadiazine for kidney disease. The Progressive Fish-culturist 13(3): 135-137. Rucker, R.R., B.J. Earp and E.J. Ordal. 1954. Infectious diseases of Pacific Salmon. Trans. Am. Fish. Soc. 83: 297-312. Scott, E.L. 1921. Sugar in the blood of the dog-fish and of the sand shark. Am. J. Physiol. 55: 349-354. Shieh-, H.S. and J.R. Maclean. 1976. Blood changes in brook trout induced by infection with Aeromonas salonicida. J. Wildlife Diseases. 12: 77-82. Simpson, W.W. 1926. The effects of asphyxia and isletectomy on the blood sugar of Myoxocephalus and Amerius. Am. J. Physiol. 77: 409-418. Snieszko, S.F. and P.J. G r i f f i n . 1955. Kidney disease in brook trout and i t s treatment. Prog. Fish-culturist. 17(1): 3-13. Snieszko, S.F. I960. Microhaematocrit as a tool in fishery research and management. U.S. Fish Wildl. Serv., Spec. Sci. Rep. - Fish. 341: 15 p. . 1974. The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish.Biol. 6: 197-208. Soivio, A. and A. Oikari. 1976. Haematological effects of stress in a teleost, Esox lucius L. J. Fish. Biol. 8: 397-411. Sprague, J.B. 1969. Measurement of pollutant toxicity to fish - I. Bioassay methods for acute toxicity. Water Res. 3: 793-821. Thomas, A.E., J.E. E l l i o t t , and J.L. Banks. 1969. Haemato-logical and chemical characteristics associated with precocious male chinook salmon fingerlings. Trans. Amer. Fish. Soc. 98: 23-26. 76 Wardle, C.S. 1 9 7 2 . The changes in blood glucose in Pleuronectes platessa following capture from the wild: a stress reaction. J. Mar. Biol. Ass. U.K. 5 2 : 6 3 5 - 6 5 1 . Wedemeyer, G.A. and J.W. Wood. 1 9 7 4 . Stress as a predis-posing factor in fish diseases. U.S. Dept. Int., Fish. Dept. Leaflet - 3 8 . 8 p. Wedemeyer, G.A. 1 9 7 0 . The role of stress in the disease resistance of fishes. Pages 30 - 3 5 in Stanislas F. Snieszko, ed. A Symposium on diseases of fishes and shellfishes. Am. Fish. Soc. Spec. Publ. No. 5 . . 1 9 7 0 . Stress of anaesthesia with MS 222 and benzocaine in rainbow trout (Salmo gairdneri) J. Fish. Res. Board Can. 2 7 : 9 0 9 - 9 1 4 . . 1 9 7 2 . Some physiological consequences of handling stress in the juvenile steelhead trout (Salmo gairdneri) and coho salmon (Oncorhynchus  kisutch). J. Fish. Res. Board Can. 29: 1 7 8 0 - 1 7 8 3 . Wood, J.W. and J. Wallis. 1 9 5 5 . Kidney disease in adult chinook salmon and i t s transmission by feeding to young chinook salmon. Oreg. Fish Comm. Res. Briefs 6 : 3 2 - 4 0 . Wood, E.M. and W.T. Yasutake. 1 9 5 5 . Histopathology of kidney disease in f i s h . Amer. J. Pathol. 3 2 ( 4 ) : 8 4 5 - 8 5 7 . Yamashita, H. 1 9 6 7 . Haematological study of a species of Rockfish - II. Changes of the moisture content of blood, specific gravity, serum protein, haematocrit value and urea nitrogen level of serum in the specimens affected by ulcers. Bull. Jap. Soc. Sci. Fish. 3 3 : 9 9 5 - 1 0 0 1 . 77a SECTION VIII APPENDICES 77 APPENDIX I SODIUM PENTACHLOROPHENATE (NaPCP) A. Preparation of stock solution of NaPCP This was identical to the procedure of Alderdice (1963) except for the fact that the amount of NaOH was doubled. B. Preparation of bioassay toxicant solutions in modified  Mariot bottles 2 ml of 5N NaOH was added to 1 1 of d i s t i l l e d water in 25 1 glass carboys. To these solutions, the appro-priate volumes of the 14.413 g/1 NaPCP stock solution described above were added and the resulting mixtures made up to 25 1 and mixed with a magnetic s t i r r e r for 5 minutes. The result-ing solutions had a pH of at least 3.5. C. Preparation of toxicant solutions for the main experiment Two concentrations of NaPCP were used in the main experiment. The test tank concentration for the intermediate level of toxicant was 0.05 (0.0039 mg/1) of the incipient 96 h L C 5 0 and 0.50 (0.039 mg/1) of this value was used for the high level of toxicant. With a constant diluent flow of 3.2 1/min and a constant toxicant flow of 0.04 l/min at the mixing funnel (Fig. IV), toxicant concentrations of 0.312 mg/1 and 3.12 mg/1 were required in the 100 1 plastic toxicant reservoirs. To achieve these concentrations, 4.0 ml of 5N NaOH was f i r s t added to 10 1 of d i s t i l l e d water in each reservoir. Then, for the intermediate toxicant level 78 reservoir, 1.08 ml of the 14.413 g/1 NaPCP solution was added to this solution. 10.8 ml of this same stock solu-tion was added to this solution in the high toxicant level reservoir. The resulting solutions in both reservoirs were then brought up to 100 1 with well water and mixed for 15 minutes. When the reservoir solutions were approximately half depleted, the levels were brought up by momentarily stopping the toxicant flow and pouring 0.54 ml of the 14.418 g/1 NaPCP stock solution with 2.0 ml of 5N NaOH into the intermediate toxicant level reservoir and 5.40 ml of the same NaECP stock solution with 2.0 ml of 5N NaOH into the high toxicant level reservoir, bringing the levels in both reservoirs back up to 100 1 while mixing and resuming the toxicant flows. The total time in which the toxicant flow was stopped was about 10 minutes. The resulting pH in the reservoirs was at least 8 .5. \ 79 APPENDIX II MICROBIOLOGICAL PROCEDURES A » Preparation of Evelyn's kidney disease media-j^ ^K D M - J - J J ) The media KDM-J--J--J- consists of Media-j- plus foetal calf serum and has the following composition: Peptone 10.00$ Yeast extract 0.05$ Cysteine - HC1 0.10$ Agar - 1.50$ Foetal calf serum in d i s t i l l e d water 20.00$ 1. Preparation of Media-j-0.313 g of cysteine-hydrochloride (Fisher Chem. Co.) was dissolved in 250 ml of d i s t i l l e d water without heat and the pH adjusted to 6.5 + 0 . 2 with 2N NaOH. 3.125 g of peptone (Difco Lab.), 0.156 g of yeast extract (Difco Lab.) and 4.688 g of agar (Difco Lab.) were then added to this solution with heat and constant sti r r i n g u n t i l the agar was completely dis-solved. This Media-j- was poured into a 500 ml screw cap bottle and sterilized in an autoclave at 15 psi and 250° F for 15 min. 2. Preparation of KDM-j.-j.-j. 62.5 ml aliquots of st e r i l e , virus screened foetal calf serum (Microcan Research Ltd., Calgary, Alta.) were stored at -20 C. At the end of the ste r i l i z a t i o n process, both Mediaj and one 62.5 ml aliquot of foetal calf serum, thawed at room temperature, were brought to 45 C in a 80 temperature controlled water bath. At the end of this equilibration procedure, a l l of the foetal calf serum was - poured into the 500 ml bottle containing 250 ml of Mediaj. The resulting KDM^j- j - was mixed thoroughly in the bottle for one minute. Approximately 15 ml of K D M ^ J J was poured into each of 21 plastic disposable petri plates on a level sur-face and l e f t for 16 h at room temperature for s o l i d i f i c a -tion and evaporation of excess moisture. B. Isolation and growth of kidney disease bacteria Where the v i a b i l i t y of the bacteria was desirable, a l l procedures were carried out using sterile techniques and at low temperatures (0-3 C). 1. Source of bacteria Viable kidney disease bacteria were isolated from a moribund, yearling pink salmon (0. gorbuscha) of length 19.0 cm and weight 118.2 g, reared at the Pacific Environ-ment Institute (W. Vancouver, B.C.) under natural light in a 4000 1 salt water tank in which flow and temperature were maintained at 30 1/min and 12 C. Gram stained kidney smears taken aseptically from a sample of moribund, fish from the same tank showed an abundance of gram-positive rods (mostly in pairs). Prior to transfer to the Pacific Environment Institute, these fish had been incubated and reared at an elevated water temperature (12 C) at the Pacific Biological Station (Nanaimo, B.C.) from eggs taken at Jones Creek, near Hope, B.C., and acclimatized to salt water. 81 2. Bacterial cultures After k i l l i n g the fish by concussion and removing the scales from one side, the whole fish was bathed in 95 percent ethanol, wiped and bathed again and f i n a l l y covered with tissue soaked with 95 percent ethanol. Using sterile techniques, a 5 -cm beveled cut was made along the mid-lateral body wall about 1 cm above the lateral line exposing a por-tion of the kidney. Approximately 0.75 g of kidney tissue was aseptically excised and dropped into a tissue homogenizer containing 15 ml of cold, sterile .saline and peptone (0.$5 and 0.1 percent respectively) solution. Eight 10-fold d i l u -tions of this original suspension were made with this same solution in st e r i l e , screw-capped test tubes. 0.1 ml of —8 the 10 suspension was spread over an area approximately 6.0 cm diameter on each of 12 sterile disposable petri plates containing KDM-J-J-J-. The 12 inoculated plates were placed on a level surface in an incubator at 15 C for 24 h to allow evapora-tion of excess moisture from the inoculum. They were then wrapped in a plastic sheet in pairs and incubated upside down for 14 days at the same temperature. The inoculated plates were examined every five days for the presence of contaminating organisms. Contaminants were 'excised using sterile techniques. After 10 days of incubation, the kidney disease bacteria started to appear in colonies on the surface of the media. However, instead of being in discrete colonies the growth was confluent and presented a ''ground glass" 6*2 appearance indicating a very heavy growth on a l l plates. The cells were harvested on the 14th day of incubation when the cells were in their log-phase of growth (T. Evelyn, per. comm.) C. Harvesting of kidney disease bacteria cells and prepara- tion of inoculum for injection into fish Kidney disease bacteria in their log-phase of growth (14 days on KDMJ-J-J at 15 C; T. Evelyn ?per. comm.) were washed1, into 25 ml of chilled sterile saline and peptone solution (0.85 and 0.1 percent respectively). The following procedures were carried out at room temperature to derive the optical density of this milky suspension of c e l l s . A l l suspensions were thoroughly agitated before aliquots were extracted for dilutions. Eight 1:1 dilutions, in the saline and peptone solution above, were made from a 3 ml aliquot of this orig-inal suspension. The optical density was determined for each dilution using the offset method on a spectrophotometer (Guilford Instr. Inc., Ohio; model 240) at 420 nm. Of these dilutions, one that gave an optical density reading between 0.20 and 0.70 was used as a reference (T. Evelyn, per. comm.) and a linear extrapolation was made to determine the optical density of the original suspension. This was done by multi-plying the dilution factor by the optical density of that reference dilution. It was found that a dilution of 1/64 gave an optical density of 0.22. Therefore, i t was 33 determined that the optical density of the original suspen-sion was 14.0 . It was found in I n i t i a l Experiment A that an optical density of 0.1 for the injection inoculum for the main experiment would be satisfactory because i t resulted in an incubation time of approximately 40 days. This duration for the main experiment was decided on the basis of the least number of desired sampling days, the size of tanks and the number and size of available fish for the experiment. Therefore, the original suspension, chilled on ice, was diluted 140 times with cold, sterile saline and peptone solution to yield 140 ml of inoculum with an opacity of 0.1 OD at 4 2 0 nm. D. Viable counts The procedures involved in determining the actual number of viable bacteria cells that were injected into each fish consisted of making dilutions of the injection inoculum (App. IIC), plating accurately measured volumes at each of these dilutions on KDMj-j--j- and counting the numbers of colonies plated (between 30 and 300; H i l l , 1903) and as-suming each colony arose from one c e l l . Once the number of viable cells per volume was known for a specific dilution, the number of viable cells per volume for the injection ino-culum was determined by multiplying the dilution factor by the number of viable cells at that dilution. Eight 10-fold dilutions of the injection inoculum were made using a cold, sterile saline and peptone ( 0 . 3 5 and 84 0.1 percent respectively) solution on ice. From each d i l u -tion, seven 25 u l aliquots of c e l l suspension were spotted onto each of five petri plates containing K D M - Q T (App. IIA). This yielded 35 replicate counts for each dilution. The plates were incubated as described above (App. IIB). Viable counts were determined on the 14th and 21st days on incu-bation from plates showing discrete colonies. 85 APPENDIX III CATEGORIZATION OF PHYSICAL CHARACTERISTICS IN RESPONSE TO KIDNEY DISEASE INFECTION Physical Characteristics no external symptoms some fl u i d in abdominal cavity alimentary canal partially empty above plus: slightly bloated abdomen slightly enlarged kidney and hind gut haemorrhaging in internal body walls (esp. at injection site bloated abdomen moderate amount of f l u i d in abdominal cavity haemorrhaging in l i v e r tissue, gonads and inter nal body walls alimentary canal empty enlargement of kidney and hind gut pale head kidney bloated abdomen as in #3 some yellow f l u i d exuding from vent moderate amount of f l u i d in abdominal cavity as haemorrhaging as in #3 alimentary canal as in #3 pale head kidney as in #3 kidney and l i v e r enlargement as in #3 small lesions on kidney bloated abdomen as in #3 excessive f l u i d in abdominal cavity few unbroken welts some yellow f l u i d exuding from vent as in #4 haemorrhaging as in #3 86 enlarged and pale spleen smokey appearance of swimbladder swollen, pale kidney - lumpy with lesions in severe cases lesions appearing on other organs (liver, spleen) #6 as in #5 plus: broken welts petechiae haemorrhaging vent internal haemorrhaging spread to other tissues -non-specific moderate autolysis X 37 APPENDIX IV Tables of mean, standard error of the mean and grand mean values for HCT, Hb, RBC, WBC, MCV, BUN, TP and GLU APPENDIX IV, TABLE I Haematocrit (HCT; %) 4 days 8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days Grand Mean r 37.08 a h 35.74 35.44 36.21 35.68 31.59 33.75 33.80 32.47 34.64 C . 6 l 3 b .756 .938 1.92 1.20 1.02 .557 .681 .996 CW F 32.94 E 1.23 28". 13 25.66 20.54 23.22 21.92 # # * 25.40 .794 .921 .904 .814 1.70 INT E 36.28 .603 30.38 1.02 34.77 .918 26.19 .811 31.80 .533 25.63 .745 34.05 1.09 23.60 1.09 34.29 .432 13.73 1.19 35.63 .696 20.13 .961 36.13 .523 34.71 .774 32.90 .676 34.51 24.12 HIGH E 34.47 .366 23.39 .366 32.42 33.45 31.43 32.53 31.03 33.62 35.30 34.33 1.06 .751 ' .545 .641 .631 .742 .445 1.12 33.19 23.39 CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h LC 5 0) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = a = b = * = 12 (two fish pooled for each measurement. ) Mean Standard Error No data due to mortality Haemoglobin (Hb; mg/100 ml) 4 days 8 days 12 days 16 days 20 days 24 days 23 days 32 days 36 days Grand Mean rt 10.23 a, 9.29 9.07 7.95 7.93 7.35 7.19 7.50 7.74 3.26 U .101b .193 .237 .165 .147 .116 .113 .237 .233 CW T? 7.93 6.43 6.63 5.23 5.39 4.13 * x x 5.99 I l l .237 .193 .196 .305 .211 .274 n 3.30 3.13 3.36 3.13 3; 63 3.54 3.23 3.20 7.67 3.32 .223 .266 .217 .332 .133 .147 .274 .217 .237 INT T? 3.33 6.63 6.13 5.02 4.13 4.93 x x 5.97 Hi .165 .176 .274 .320 .277 .199 r< 3.33 3.34 3.65 7.23 7.42 7.35 7.07 7.27 6.30 7.61 L> .349 .303 .176 .167 .292 .165 .139 .130 .199 HIGH F. 7.02 * * * * * x x 7.02 Ill .346 CW = Clean Water (0 x 96 h LC 5 0) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h LC 5 Q) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = a = b = * = 12 (two fish pooled for each measurement. ) Mean Standard Error No data due to mortality APPENDIX IV, TABLE III Red blood c e l l count (RBC; no. of cells/mmVlO,000) 4 days 8 days 12 days 16 days 20 days 24 days 2 8 days 32 days 36 days Grand Mean CW 1 4 7 . 8 3 ? 1 4 7 . 8 3 1 4 7 . 4 2 1 5 9 . 1 7 6 . 8 2 B 6 . 4 7 7 . 8 2 5 . 4 8 1 5 1 . 0 0 1 5 3 . 0 7 6.46 6 . 3 5 1 4 9 . 1 7 1 5 3 . 7 1 1 4 9 . 1 4 1 5 0 . 9 3 6 . 1 4 4 . 9 5 6 . 1 8 E 1 2 2 . 9 2 5 . 2 2 1 1 7 . 3 3 6 . 7 7 9 7 . 1 7 4 . 7 5 9 2 . 0 0 4.84 9 9 . 4 2 4 . 3 7 7 3 . 4 2 5.46 * * * 1 0 0 . 3 8 C INT E 1 4 9 . 4 2 5 . 4 5 1 2 9 . 5 0 6 . 9 0 137.67 6.76 9 4 . 3 4 8 . 0 6 146.67 8.26 94 .67 5 . 1 8 1 4 7 . 1 7 8 . 0 2 9 4 . 9 2 6 . 4 4 163 .58 3 . 2 5 7 1 . 7 5 5 . 7 8 1 5 1 . 9 2 7 . 3 7 7 1 . 5 0 5 . 5 8 1 5 3 . 1 7 5 . 1 9 * 1 4 2 . 8 6 7 . 3 7 * 1 5 2 . 7 2 7 . 7 8 * 1 4 9 . 4 6 9 2 . 7 8 C HIGH E 1 3 9 . 6 7 8 . 0 8 1 4 3 . 7 5 6 . 9 1 1 5 0 . 3 3 6.18 136.OO 6 . 0 4 1 5 6 . 5 8 5 . 1 2 1 4 8 . 8 3 3 . 7 5 1 4 8 . 0 8 8 . 4 3 1:52.43 4 . 3 8 162 .86 4 . 9 2 1 4 8 . 7 3 1 1 3 . 1 7 6 . 9 3 * * * # * * * * 1 1 3 . 1 7 CW = Clean Water ( 0 x 96 h LC 5 0) INT = Intermediate Level NaPCP ( 0 . 0 5 x 96 h LC 5 0) HIGH = High Level NaPCP ( 0 . 5 x 96 h L C ^ Q ) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = a = b = * _ 1 2 (two fish pooled for each measurement. ) Mean Standard Error No data due to mortality APPENDIX IV, TABLE IV Total white blood c e l l count (WBC; no. of cells/mm 3 /500) 4 days 3 days 1 2 days 16 days 20 days 2 4 days 2 3 days ; 32 days 36 days Grand Mean CW C .352° 3 . 1 7 .600 4 . 5 0 .621 3 . 4 2 .621 4 . 1 7 . 3 7 8 4 . 3 3 . 7 9 1 3.67 .667 4 . 5 7 . 2 2 3 4 . 4 3 ^ . 4 0 4 3 . 3 9 E 1 . 5 8 . 3 7 8 2 . 0 0 . 4 0 7 1 . 8 3 . . 5 2 8 2 . 3 3 . 4 5 0 3 . 0 0 .626 4 . 4 2 1 . 0 4 x x x 2 . 5 3 INT C E 4 . 2 5 . 4 9 4 2 . 3 3 .396 4.67 . 7 8 2 1 . 5 8 .260 4.42 . 7 8 2 1.92 . 5 2 5 4 . 0 0 . 7 3 9 3 . 5 8 .667 4 . 0 3 . 5 5 7 5.67 1.06 4 . 5 3 . 3 5 7 7 . 4 2 . 6 5 3 3 . 2 5 . 7 3 9 X 2 . 0 0 . 4 0 7 x 2 . 4 3 . 4 9 7 * 3 . 7 4 3 . 7 5 HIGH C E 3 . 1 7 . 4 9 1 1 . 5 8 . 3 5 8 4 . 4 2 .621 x 3 . 9 2 . 5 4 3 x 3 . 3 3 . 6 5 5 * 4 . 0 3 .794 4 . 4 2 .932 x 2.50 .436 X 2 . 0 0 .647 x 2 . 5 0 .471 X 3 . 3 7 1 . 5 3 CW = Clean Water ( 0 x 9 6 h LC^) INT = Intermediate Level NaPCP ( 0 . 0 5 x 9 6 h L C ^ Q ) HIGH = High Level NaPCP ( 0 . 5 x 9 6 h LC 5 0) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = a = b = x _ 12 (two fish pooled for each measurement. ) Mean Standard Error No data due to mortality APPENDIX IV, TABLE V Mean c e l l volume (MCV; u 3) 4 days 8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days Grand Mean CW C 256.03^ 11.42° 248.79 15.49 246.71 12.89 230.23 14.44 240.85 13.19 205.59 12.50 229.93 8.39 254.64 23.04 220.94 10.12 237.03 E 271.60 12.80 245.87 11.93 273.25 18.74 228.28 12.81 236.27 9.13 - 310.02 24.86 * x x 260.33 INT C E 247.13 11.70 239.56 11.11 256.64 9.45 298.55 26.58 223.66 11.93 277.34 12.15 236.05 10.51 257.19 15.17 210.70 5.51 271.13 16.08 239.57 10.23 292.32 15.81 239.15 9.53 x 221.41 5.94 x 220.84 11.07 x 232.79 272.63 HIGH C E 254.16 13.12 266.00 18.28 231.28 13.63 * 227.35 7.02 * 235.81 10.27 x 203.09 11.21 * 209.89 5.91 x 234.15 12.16 x 233.23 6.29 x 212.79 9.38 x 226.36 266.00 CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h L C 5 0 ) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n a b x 12 (two f i s h pooled for each measurement. ) Mean Standard Error No data due to mortality APPENDIX IV, TABLE VI Blood urea nitrogen (BUN; mg/100 ml) 4 days 8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days Grand Mean < n 8.38a 4.73 4.92 5.38 6.23 5.41 4.78 3.09 3.87 5.20 \J .595 .517 .814 .433 .499 .424 .753 .329 .375 cw 4.80 TT* 4.18 3.02 3.85 6.57 4.54 6.75 x X X Cl .167 .199 .370 .782 .318 .629 rt 10.91 8.08 11.16 14.36 10.04 5.59 3.90 2.89 3.43 7.81 U 1.11 .849 .759 2.05 1.06 .664 .159 .338 .257 INT 3.62 T? 5.25 2.83 2.18 3.52 3.59 4.34 x X X Ci .^344 .199 .335 .176 .277 .196 rt 8.73 4.24 5.13 3.98 9.79 6.38 7.52 3.46 3.40 5.45 .612 .124 .606 .367 1.37 .710 .473 .476 .306 HIGH 6.57 V. 6.57 x x x x x X X X ill .453 CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h LC 5 Q) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n a b x 12 (two fish pooled for each measurement. ) Mean Standard Error No data due to mortality APPENDIX I V . TABLE V I I T o t a l p r o t e i n ( T P ; g/100 m l ) 4 d a y s 8 d a y s 12 days 16 d a y s 20 days 24 d a y s 28 d a y s 32 d a y s 36 days G r a n d Mean 3 . 4 9 a h 3 . 7 9 3 . 8 6 3 .13 4.03 4.31 3 . 9 3 3 . 7 6 3 . 7 6 3 . 7 3 L. . 1 8 5 b .136 . 1 2 1 . 110 .179 .156 . 1 7 9 .404 .113 CW 2 . 8 6 2.61 3 . 4 7 4 . 3 4 2.30 2 . 7 1 x x x 3 . 0 5 £» . 1 4 7 . 208 .225 . 147 . 217 .156 ri 4 . 7 3 4 . 1 7 4 . 1 8 4 . 7 3 4 . 5 9 3 . 6 8 3 . 7 7 3 . 8 3 3 . 4 0 4 . 1 2 0 . 245 .245 .294 . 2 4 0 .364 .225 . 2 6 0 . 6 0 0 .136 INT 2 . 3 9 TT* 3 . 1 8 2 . 6 0 2 . 7 7 3 . 0 1 3.16 2 . 6 4 x x x £j .153 . 211 .205 .326 .193 . 297 rt 2 . 9 3 3 . 1 5 3.23 3 .33 3 . 1 8 3 . 7 7 3 . 9 6 3 . 1 7 4 . 0 4 3 . 4 2 U . 2 5 1 . 1 9 1 .245 . 251 .124 . 196 . 1 4 7 .508 .233 HIGH 2 . 0 9 T? 2 . 0 9 # * * X x x x Hi . 271 CW = C l e a n W a t e r (0 x 96 h L C c n ) INT = I n t e r m e d i a t e L e v e l NaPCP ( 0 . 0 5 x 96 h L C ^ Q ) HIGH = H i g h L e v e l NaPCP ( 0 . 5 x 96 h L C ^ Q ) C = U n i n f e c t e d C o n t r o l Group E = K i d n e y D i s e a s e I n f e c t e d E x p e r i m e n t a l Group n = a = b = x = 12 ( two f i s h p o o l e d f o r each m e a s u r e m e n t . ) Mean S t a n d a r d E r r o r No d a t a due t o m o r t a l i t y Glucose (GLU; mg/100 ml) 4 days 8 days 1 2 days 16 days 2 0 days 24 days 2 8 days 3 2 days 36 days Grand Mean GW C 6 0 . 8 1 * 2 . 7 9 6 2 . 3 8 2 . 4 6 5 4 . 4 0 2 . 4 9 6 0 . 4 3 3 . 3 3 5 6 . 5 4 2 . 6 6 5 7 . 0 4 1 . 9 4 5 9 . 2 5 3 . 7 7 6 4 . 2 9 1 . 4 9 6 2 . 0 7 1 . 4 3 5 9 . 6 9 E 5 6 . 4 6 1 . 4 9 3 5 . 5 8 2 . 4 5 3 3 . 9 2 1 . 3 9 3 8 . 6 3 2 . 2 2 3 0 . 2 1 1 . 6 5 3 7 . 1 7 2 . 2 8 * * * 3 8 . 6 6 INT C E 7 6 . 7 9 3 . 5 5 6 0 . 0 3 2 . 5 0 5 8 . 1 7 2 . 3 5 4 3 . 5 0 2 . 1 8 5 2 . 0 8 2 . 9 6 3 5 . 5 0 2 . 1 0 6 2 . 0 8 2 . 5 8 3 5 . 4 2 2 . 1 2 6 1 . 5 0 1 . 8 7 3 8 . 5 4 2 . 5 2 7 9 . 2 9 2 . 3 2 3 8 . 7 9 3 . 3 7 5 2 . 9 2 3 . 4 5 # 6 8 . 5 7 4 . 5 5 * 1 0 1 . 0 7 6 . 3 9 * 6 8 . 0 5 4 1 . 9 7 HIGH C E 5 3 . 0 0 5 . 3 8 4 1 . 0 4 3 . 0 0 4 6 . 0 0 3 . 2 4 * 4 5 . 9 2 2 . 1 7 # 3 7 . 4 6 . 9 5 0 6 2 . 2 5 2 . 1 6 5 5 . 6 7 3 . 4 0 * 5 7 . 9 2 2 . 1 3 * 7 9 . 9 3 3 . 4 8 4 9 . 9 3 3 . 2 7 * 5 4 . 2 3 4 1 . 0 4 CW = Clean Water ( 0 x 9 6 h LC C A) INT = Intermediate Level NaPCP ( 0 . 0 5 x 9 6 h L C ^ Q ) HIGH = High Level NaPCP ( 0 . 5 x 9 6 h LC 5 Q ) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = a = b = * _ 1 2 (two fish pooled for each measurement. ) Mean Standard Error No data due to mortality 9 6 ) APPENDIX V Results for Mean Corpuscular Haemoglobin Concentration and Mean Cell Haemoglobin 97 The results for MCHC and MCH were highly variable in a l l groups over the entire sampling period. In some cases, these results seemed to even contradict those patterns that would be expected from the trends in the values used to de-rive the MCHC and MCH values. For example, experimental fish in group IE showed i n i t i a l l y lower, but not significant, MCHC values relative to sham injected controls in group IC on the f i r s t two sampling days (Tl and T2; App. V Fig. I ) . Thereafter, the MCHC values in group IE fish increased and remained above control f i s h values of group IC u n t i l the sixth sampling day (T6). The MCHC values of group 2E declined, relative to group 2C values, from being significantly higher on the f i r s t two sampling days (Tl and T2) to being only slightly lower (P greater than .05) than group 2C values on the fourth sampling day ( T 4 ) . After the decline by time T 4 , the MCHC for experimental fish increased u n t i l they were significantly higher than the 2C group values on the sixth sampling day. No significant difference was observed be-tween group 3E and 3C MCHC values on the f i r s t sampling day (Tl). The MCH values in group IE fish were found to be significantly lower on the f i r s t two sampling days (Tl and T2; App. V Fig. II) relative to group IC values on the same days. The IE group values then increased u n t i l they were s i g n i f i -cantly higher than the values of fish in group IC on the fourth sampling day ( T 4 ) . After the MCH values for group IE dropped, once again, to being significantly lower than the. control group value by the sixth sampling day (T6). The 98 The reverse trend seemed to occur in fish of groups 2E and 2C. The MCH values of group 2E started out significantly-higher than group 2C values on the f i r s t two sampling days (TI and T2) after which they dropped to be significantly lower than group 2C values by the fourth sampling day (T4). Then, the group 2E values rose to a slightly higher, but not significant, level relative to 2C values on the sixth sampling day (T6). N O significant difference occurred between MCH values of fish in groups 3E and 3C on the f i r s t sampling day (TI). Due to the marked fluctuations and inconsistent trends in the mean corpuscular haemoglobin concentration and in the mean cellular haemoglobin values in a l l groups of fish over the experimental period, no attempt was made to draw any conclusions from these results. APPENDIX V, TABLE I Mean corpuscular haemoglobin concentration (MCHC; pg) 4 days 8 days 12 days 16 days 2 0 days 2 4 days 2 8 days 3 2 days 3 6 days Grand Mean n 7 0 . 9 9 ? 6 4 . 2 5 6 1 . 2 3 5 0 . 4 3 5 3 . 7 7 4 7 . 6 4 4 3 . 9 4 5 3 . 4 0 5 2 . 6 4 5 5 . 9 2 2 . 9 9 ° 3 . 1 5 3 . 4 3 1 . 5 6 2 . 1 3 2 . 2 9 1 . 7 3 1 . 3 3 2 . 5 9 CW TP 6 5 . 8 7 6 2 . 3 0 7 0 . 0 6 5 7 . 9 3 5 4 . 6 6 5 3 . 0 5 x x x 6 1 . 4 3 Hi 2 . 9 2 3 . 0 0 4 . 0 4 2 . 3 1 1 . 3 5 2 . 7 3 r> 5 6 . 1 0 6 0 . 2 2 6 2 . 2 2 5 6 . 5 5 5 3 . 3 4 5 7 . 5 0 5 4 . 2 6 5 2 . 7 4 5 1 . 1 3 ' 5 6 . 0 0 O 3 . 4 6 3 . 3 4 3 . 4 5 2 . 4 9 1 . 3 9 2 . 5 1 2 . 3 3 1 . 1 5 2 . 0 1 INT TP 7 0 . 4 6 7 5 . 1 5 6 5 . 9 5 5 3 . 3 3 5 9 . 3 4 7 1 . 7 4 x x x 6 6 . 0 3 Cl 3 . 6 4 5 . 1 6 1 . 7 3 1 . 3 0 3 . 3 9 3 . 3 9 r> 6 0 . 9 0 5 3 . 3 7 5 3 . 5 3 5 1 . 3 2 4 7 . 6 9 4 9 . 5 2 4 9 . 1 3 4 3 . 0 3 4 2 . 1 4 5 1 . 3 5 O 1 . 9 4 2 . 4 3 2 . 4 2 4 . 2 1 1 . 9 7 1 . 2 0 2 . 5 4 1 . 3 7 1 . 7 6 HIGH 6 4 . 7 1 TP 6 4 . 7 1 x * * * * x x Hi 5 . 2 0 CW = Clean Water ( 0 x 9 6 h L C ^ Q ) INT = Intermediate Level NaPCP ( 0 . 0 5 x 9 6 h L C ^ Q ) HIGH = High Level NaPCP ( 0 . 5 x 9 6 h LC,n) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group n = a = b = x — 1 2 (two fi s h pooled for each measurement. ) Mean Standard Error No data due to mortality APPENDIX V, TABLE I I Mean c e l l u l a r haemoglobin (MCH; $) 4 days 3 days 12 days .16 days 2 0 days 24 days 28 days 3 2 days 3 6 days Grand Mean n 27.81 a, 26.07 25.64 22.49 22.70 23.56 21.34 21.76 23.85 2 3 . 9 6 O .401° .502 .970 1.02 . 9 9 3 .924 .315 .941 . 3 9 6 CW 23.76 24.33 23.10 26.11 25.90 23.44 19.66 x x x E .499 . 5 4 6 .987 1.43 1.04 1.44 rt 22.91 2 3 . 6 3 27.93 24.12 25.35 24.07 22.74 2 4 . 2 4 2 3 . 3 0 24.25 0 . 6 3 2 .722 .722 .889 .341 .551 .580 . 3 5 2 . 4 5 6 INT 2 4 . 6 9 TT* 29.47 25.56 24.07 21 . 2 3 22 . 0 3 - 24.80 x x x C l .875 .756 .707 .979 .482 1.09 r> 24 . 3 0 25.98 25.95 2 3 . 0 3 22.89 23.72 21.08 20 . 6 4 19.91 23.05 .771 1.09 .583 .494 1.10 .505 .473 . 4 6 8 .580 HIGH F. 2 4 . 4 2 * * X x x x x 2 4 . 4 2 Ill 1.19 CW = Clean Water (0 x 9 6 h L C ^ Q ) INT = Intermediate L e v e l NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Le v e l NaPCP (0.5 x 9 6 h L C ^ Q ) C = Uninfected C o n t r o l Group E = Kidney Disease Infected Experimental Group n = a = b = x -12 (two f i s h pooled f o r each measurement. ) Mean Standard E r r o r No data due t o m o r t a l i t y 101a Appendix V, Figures I and II Graphs of the response of MCHC and MCH values in the different groups of fish to the experimental treat-ments, bacterial kidney disease infection and NaPCP exposure, over the sampling period. Each point repre-sents a mean + 1.96 standard error of the mean (n=12). Responses in uninfected control fish are designated by solid lines and kidney disease infected fish by broken lines, a, b and c represent clean water (0 x 96 h L C ^ Q ) , intermediate level of NaPCP exposure (0.05 x 96 h L C ^ Q ) , and high level of NaPCP exposure (0.5 x 96 h LC^Q) res-pectively. T l represents the f i r s t sampling day, four days after beginning toxicant exposure. 2 to 9 repre-sent subsequent sampling days. Symbols on top of each sampling day designate s t a t i s t i c a l significance (P=0.05), by Scheffe's test, between means of different groups of fish: * = between control and experimental f i s h , 1 = in a, 2 = in b, 3 = in c; H = in control fish groups, A = between a and b, B = between b and c, C = between a and c; D = in kidney disease infected f i s h . Appendix V, Figure I Mean Corpuscular Haemo-globin Concentration Appendix V, Figure II Mean Cellular Haemoglobin 101 Appendix V, Figure I o HBC HB 80_ 60 40L 12 HB HBC E-i a w o a o o m o a w ac •CE! < J=> O CO !=> OH Ct! O O b 85_ 65 45 c 75_ 55 35 TI 2 3 4 5 6 7 8 9 SAMPLING PERIOD - 4 day in t e r v a l s 102 Appendix V, Figure II a 29> 12 12 HAC DAB °A 2 12. 2 1 D. HBC HB E-H • S E-H O o PQ O hi a pc; < hi w o b 31 27L 23 19 c 28 24 20 I 1 I L J L J L Tl 2 3 4 5 6 7 8 SAMPLING PERIOD - k day intervals 103a Appendix V, Figures III and IV Histograms showing grand means and means of absolute differences between control and experi-mental fish for the different groups of fi s h . Sam-ple sizes used to derive these values are indicated on top of bars. Appendix V, Figure III Mean Corpuscular Haemo-globin Concentration Appendix V, Figure IV Mean Cellular Haemoglobin MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION•-grand means (pg) i—1 o I L_ L j O ^ o * means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and e x p e r i m e n a l f i s h . MEAN CELLULAR HAEMOGLOBIN CONTENT -grand means (%) o 00 • o O o O O 1  ! 1 i I o NO fD J o * i mm J la fD CO n co fD CO * O 3 -N3 CO * means of absolute differences between control and experimental f i s h . * 105 APPENDIX VI PHOTOGRAPHS OF APPARATUS USED IN BIOASSAYS AND THE MAIN EXPERIMENT 106 

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