UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Gas bubble trauma in fish Fidler, Larry E. 1988

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1988_A1 F52.pdf [ 19.24MB ]
Metadata
JSON: 831-1.0097966.json
JSON-LD: 831-1.0097966-ld.json
RDF/XML (Pretty): 831-1.0097966-rdf.xml
RDF/JSON: 831-1.0097966-rdf.json
Turtle: 831-1.0097966-turtle.txt
N-Triples: 831-1.0097966-rdf-ntriples.txt
Original Record: 831-1.0097966-source.json
Full Text
831-1.0097966-fulltext.txt
Citation
831-1.0097966.ris

Full Text

GAS BUBBLE TRAUMA IN FISH by LARRY E. FIDLER B.Sc., The Pennsylvania State University, 1960 M . S c T h e University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accep t this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA July 31,1988 ©Larry E. Fidler, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of TZ&OLOG-"/ The University of British Columbia Vancouver, Canada Date aCTZA&Z DE-6 (2/88) i i ABSTRACT Fish exposed to gas supersaturated water often experience a form of stress known as Gas Bubble Trauma (GBT). GBT is an acute condition involving various forms of bubble growth both internal and external to the animal. Theoretical models are developed which establish thresholds for bubble growth. These models apply to: 1. ) Bubble growth in the vascular systems of fish. 2. ) Bubble growth in the environmental water that can occur in the buccal cavity and between gill lamella. 4.) Overinflation of the swimbladder. 3. ) Sub-dermal bubbles that occur on external skin surfaces such as the opercular flaps, between fin rays and in the lining of the mouth. In order to develop the models for general use, it was necessary to establish the effective size of nucleation sites and other physiological parameters contained in the bubble growth threshold equations. This was accomplished through a review of data from the scientific literature and a two phase experimental program. The literature review resulted in the compilation of a database containing over 1000 records of supersaturation data on salmonids. Various filters based on length, species, total gas pressure (TGP), partial pressure of oxygen (PO2) and other criteria were applied to the database. The filtering operations established the existence of GBT mortality thresholds and identified relationships between other experimental parameters. The results of this analysis suggest that a lower threshold occurs at a water TGP of 1.10 Atms. and a higher threshold occurs at 1.15 to 1.18 Atms. However, it was not established that the apparent mortality thresholds correspond to thresholds for bubble growth predicted by the theoretical models. To make this correlation, a preliminary experimental study examined the physiological response of fish exposed to supersaturated water. It was found that arterial pC>2, hematocrit and blood pressure yield unique responses to bubble growth over specific ranges of water TGP. The results of these experiments also indicate that the lower mortality threshold of the database analyses is associated with a combination of sub-dermal bubble growth in the mouth and extracorporeal bubbles growing between gill lamella. The second phase of experimental study included surveys of blood p02, hematocrit and pH along with microscopic studies of intravascular and extracorporeal bubble growth in gills. The results of these experiments confirm the source of mortality for the lower threshold at a water TGP of 1.1 Atms. In addition, the data demonstrate that the upper TGP threshold of 1.15 to 1.18 Atms. of the database analysis corresponds to the threshold for intravascular bubble growth. The results further confirm that, as predicted by the theoretical model, intravascular bubble growth thresholds are dependent on water p02 • Combining the results of the database analysis and the experimental studies permitted the effective size of nucleation sites responsible for bubble growth to be back calculated from the theoretical equations. This completed the development of the bubble growth threshold equations. The equations can now be used to predict thresholds for the various forms of bubble growth and mortality that occur in fish i v exposed to supersaturated water. The experimental results also provide valuable information regarding the physiological response of fish to gas supersaturated water. TABLE OF CONTENTS PAGE Abstract ii List of Tables ix List of Figures x Acknowledgements xvi List of Abbreviations xvii 1.0 Introduction 1 1.1 Dissolved Gas Supersaturation 1 1.2 Causes of Supersaturation 3 1.3 Symptoms of Gas Bubble Trauma 5 1.4 Reporting of Dissolved Gas Tensions 5 1.5 Experimental Studies 6 1.6 Analytical Models 9 1.61 Time to 50% Mortality 9 1.62 Bubble Growth Thresholds 9 2.0 Study Definition 13 3.0 Theoretical Studies 15 3.1 Introduction 15 3.2 Thresholds for Bubble Growth 15 3.2.1 Vascular System Bubble Growth 16 3.2.2 Swimbladder Overinflation 18 3.2.3 Water Bubbles 18 3.3 Background and Methods 19 3.3.1 Equation Modification 19 3.3.2 Gill Oxygen Uptake Ratio 21 3.3.3 Arterial Blood Pressure 3.3.4 Critical Nuclei Radius 3.3.5 Effective Nuclei Radius 3.3.6 Subdermal Bubble Growth 3.3.7 Bubble Growth in an Open System 3.3.8 Bubble Growth in a Closed System 3.4 Results 3.4.1 Threshold Equations 3.4.2 Oxygen Uptake Ratio 3.4.3 Bubble Growth in an Open System 3.4.4 Bubble Growth in a Closed System 3.5 Discussion 3.5.1 Oxygen Uptake Ratio 3.5.2 Bubble Growth Threshold Equations 3.5.3 Bubble Growth Rate 3.6 Theoretical Summary 4.0 Gas Bubble Trauma Database 4.1 Methods and Materials 4.1.1 Data Sources 4.1.2 Methods of Analysis 4.2 Results 4.2.1 Preliminary Filtering 4.2.2 Unique Data Sets 4.2.3 Effect of Fish Size 4.2.4 Effect of Fish Species 4.2.5 Effects of Water Oxygen Tension 4.2.6 Compensation Depth 80 4.3 Discussion 85 Experimental Studies 93 5.1 Introduction 93 5.1.1 Phase I Experiments 95 5.1.2 Phase II Experiments 95 5.2 Experimental Materials and Methods 95 5.2.1 Experimental Apparatus and Water Measurements 96 5.2.2 Computerized Data Acquisition System 100 5.2.3 Experimental Animals and Surgery Procedures 101 5.2.4 Physiological Parameters 103 5.2.5 Phase I Experiments 105 5.2.6 Phase II Experiments 106 5.3 Phase I Experimental Results 111 5.3.1 General Observations 111 5.3.2 Response of Individual Fish 113 5.4 Discussion of Phase I Results 130 5.4.1 General Response to Supersaturation 130 5.4.2 GBT Between a TGP of 1.1 and 1.15 Atms. 131 5.4.3 GBT Between a TGP of 1.15 and 1.25 Atms. 134 5.5 Phase I Conclusions 142 5.6 Phase II Experimental Results 144 5.6.1 Series 1 through 5 Experiments 144 5.6.2 Series 6 Experiments 172 5.6.3 Series 7 Experiments 5.6.4 Series A, B. C and Series 4 Experiments 5.6.5 Other Results 5.7 Discussion of Phase II Experimental Results 5.7.1 Extravascular and Subdermal Bubble Growth Thresholds 5.7.2 Intravascular Bubble Growth Thresholds 5.7.3 Bubble Growth at Low TGP Levels 5.7.4 Bubble Growth at High TGP Levels 5.7.5 Time to Mortality 5.7.6 Response to Hypoxia 6.0 Concluding Discussion References Appendices LIST OF TABLES Table I: Nomenclature. Table II: Abbreviations for Severity of Symptoms. Table III: Vascular System Bubble Survey Series A, B, C & 4 LIST OF FIGURES FIGURE NO. TITLE x PAGE FIGURE 1: Threshold Nuclei Radius Versus 26 Blood Pressure. FIGURE 2: Threshold Nuclei Radius Versus Water TGP. 28 FIGURE 3: Effective Radius of Nucleation Sites in 29 Vascular Bubble Growth Equation. FIGURE 4: Theoretical Water Total Gas pressure 37 Thresholds. FIGURE 5: Ratio of Arterial to Water p 0 2 for 39 Rainbow Trout. FIGURE 6: Variation in Arterial Bubble Growth 41 Thresholds Versus Oxygen Uptake Ratio. FIGURE 7: Bubble Radius Versus Time for Bubble 42 Growth in an Open System. FIGURE 8: Bubble Growth in a Closed System, 44 TGP = 1.17 Atms. FIGURE 9: Bubble Growth in a Closed System, 45 TGP = 1.316 Atms. FIGURE 10: Swimbladder Inflation Thresholds. 48 FIGURE 11: Time to Mortality for all Fish 58 in Database. FIGURE 12: Time to Mortality Versus Water TGP 59 Mortality Range = 20 - 70%. FIGURE 13: Time to Mortality Versus Fish Length TGP = 1.08- 1.5 Atms. FIGURE 14: Time to Mortality Versus Fish Length TGP = 1.08- 1.15 Atms. FIGURE 15: Time to Mortality versus Fish Length TGP = 1.15- 1.20 Atms. FIGURE 16: Time to Mortality versus Fish Length TGP = 1.20- 1.50 Atms. FIGURE 17: Time to Mortality Versus Water TGP for Fish Less Than 50 mm. in Length. FIGURE 18: Time to Mortality Versus Water TGP for Chinook Salmon Over 50 mm. in Length. FIGURE 19: Time to Mortality Versus Water TGP for Sockeye Salmon Over 50 mm in Length. FIGURE 20: Time to Mortality Versus Water TGP for Coho Salmon Over 50 mm. in Length. FIGURE 21: Time to Mortality Versus Water TGP for Steelhead Trout Over 50 mm. in Length. FIGURE 22: Time to Mortality Versus Water TGP for Cutthroat Trout Over 50 mm. in Length. FIGURE 23: Time to Mortality Versus Water TGP for Fish Less Than 50 mm. FIGURE 24: Time to Mortality Versus Water TGP for Fish Greater Than 50 mm. x i i 76 FIGURE 25: Time to 50 % Mortality Versus Water p 0 2 78 for Steelhead Trout. FIGURE 26: Time to Mortality Versus Water p 0 2 79 for Coho Salmon, TGP = 1.193 Atms. FIGURE 27a: Time to Mortality Versus Water TGP for 82 Fish Less Than 50 mm.; Depth Correction. FIGURE 27b: Time to Mortality Versus Water TGP for 82 Fish Greater Than 50 mm.; Depth Correction. FIGURE 28: Time to Mortality Versus Water TGP for 83 Chinook, Coho and Steelhead Trout. FIGURE 29: Time to 50 % Mortality Versus Water TGP 84 With and Without Depth Correction. FIGURE 30: Model 1 of Schnute and Jensen 1986 87 FIGURE 31 : Model 15 of Schnute and Jensen 1986 88 FIGURE 32: TGP Thresholds for Bubble Growth in 91 Arterial Blood. FIGURE 33: Experimental Apparatus 98 FIGURE 34: Live Box 99 FIGURE 35: Fish in van Dam Respiration Chamber 110 FIGURE 36: Fish 9 Dorsal Aorta Blood Pressure. 115 FIGURE 37: Fish 22; Pre-exposure Blood Pressure Blood Pressure after 86 Hours. FIGURE 38: Fish 22; Blood Pressure after 200 Hours. Blood Pressure after 256 Hours. FIGURE 39: Fish 11; Arterial Blood pressure. Pre-exposure, 75 Hours and 105 Hours. FIGURE 40: Fish 11; Arterial Blood pressure. Pre-exposure, 115, 117 and 118 Hours. FIGURE 41: Fish 17; Pre-exposure Blood Pressure and at 10 Hours. FIGURE 42: Fish 17; Blood Pressure After 49 Hours and 56 Hours. FIGURE 43: Fish 6; Arterial Blood Pressure. FIGURE 44: Fish 27; Pre-exposure Blood Pressure and After 7 Hours. FIGURE 45: Fish 27; Blood Pressure After 12 Hours. FIGURE 46: Time to Death as a Function of Water TGP. FIGURE 47: Arterial pG^, Series 1. FIGURE 48: Arterial Hematocrit, Series 1. Arterial pH, Series 1. FIGURE 49: Severity of Symptoms at Death, Series 1. FIGURE 50: Arterial p 0 2 , Series 3. FIGURE 51: Arterial Hematocrit, Series 3. Arterial pH, Series 3. FIGURE 52: Severity of Symptoms at Death, Series 3. FIGURE 53: Extracorporeal Bubbles in Gills. FIGURE 54: Subdermal Bubbles in Mouth, View 1. FIGURE 55: Subdermal Bubbles in Mouth, View 2. FIGURE 56: Opercular Bubbles. FIGURE 57: Opercular Bubbles. FIGURE 58: Bubbles in Dorsal Fins. FIGURE 59: Bubbles in Caudal Fins. FIGURE 60: Arterial p 0 2 , Series 4. FIGURE 61: Arterial Hematocrit, Series 4. Arterial pH, Series 4. FIGURE 62: Severity of Symptoms at Death, Series 4. FIGURE 63 a, b, c & d: Vascular Bubbles in Gills. FIGURE 64 a, b, c & d: Vascular Bubbles in Gills. FIGURE 65: Arterial p 0 2 , Series 2. FIGURE 66: Arterial Hematocrit, Series 2. Arterial pH, Series 2. FIGURE 67: Severity of Symptoms at Death, Series 2. FIGURE 68: Arterial p 0 2 , Series 5. FIGURE 69: Arterial Hematocrit, Series 5. Arterial pH, Series 5. FIGURE 70: Severity of Symptoms at Death, Series 5. FIGURE 71: Adrenaline Variation with Time, Experimental Series 6. FIGURE 72: Noradrenaline Variation with Time, Experimental Series 6. FIGURE 73: Ventilation Volume and Frequency Versus Time, Experimental Series 7. FIGURE 74: Time to Mortality Versus Water p 0 2 for Constant TGP. FIGURE 75: TGP Thresholds for Bubble Growth in Arterial Blood. FIGURE 76: Bubble Growth Thresholds as a Function of Water p 0 2 xvi ACKNOWLEDGEMENTS I would like to thank Dr. Dave Randall for his guidance in this work as well as for his stimulating conversations regarding matters scientific and otherwise. I also wish to acknowledge the many helpful and enlightening discussions with Dr. Don Alderdice and John Jensen of the Pacific Biological Station and Dr. Bob White of Montana State University. The work described in this thesis was supported by a grant from the Canada Department of Fisheries and Oceans Pacific Biological Station, the NSERC grant of Dr. Dave Randall and a research assistantship at Montana State University sponsored by the U.S. Bureau of Land Management. MathCad is a registered trade name of Mathsoft, Inc. Labtech Notebook is a registered trade name of Laboratory Technologies Corporation. Lotus 123 is a registered trade name of Lotus Development Corporation. Reflex and Turbo Basic are registered trade names of Borland International Corporation. x v i i L I S T O F A B B R E V I A T I O N S GBT = Gas Bubble Trauma. TGP = Total Gas Pressure = p 0 2 + p N 2 + p H 2 0 p 0 2 = partial pressure of dissolved oxygen in environmental water. p N 2 = partial pressure of dissolved nitrogen in environmental water. p H 2 0 = vapor pressure of water. POb = P a r t i a l pressure of oxygen in bubble. PNb = P a r t i a ' pressure of nitrogen in bubble. POp = P a r t ' a ' pressure of oxygen in plasma. PNp = P a r t i a ' pressure of nitrogen in plasma. r 0 = the radius of the critical nucleation sites from which bubble growth begins (environmental water or vascular system). K = H 0 k 0 / H N - k N where, H Q = Henrys constant for oxygen in fish blood. Hjg = Henrys constant for nitrogen in fish blood, kfg = mass transfer coefficient for nitrogen, kg = mass transfer coefficient for oxygen. D Q = coefficient of diffusion for oxygen in fish blood. Djyj = coefficient of diffusion for nitrogen in fish blood. T = temperature in degrees C . o = surface tension of water or fish blood. F = oxygen uptake ratio across gills = p 0 2 (Arterial Blood)/p0 2 (Water). P = Patm. + p-g-h + Ps. Patm = atmospheric pressure. Ps = system pressure where bubble is growing, o = density of water, h = depth offish in water column. k 0 = mass transfer coefficient for oxygen. k n = mass transfer coefficient for nitrogen. R' = gas constant. C = compliance. Ro = effective nucleus radius. r = radius of growing bubble. Vg = volume of growing bubble. N = number of bubbles growing in vascular system. PQ A = T O T A L 9 a s pressure of gas A in water. g = gravitational constant. 1 1.0 INTRODUCTION Gas Bubble Trauma (GBT) is a condition that arises when the water in which fish live becomes supersaturated with dissolved atmospheric gases. The condition, first described by Robert Boyle (1670), can produce a variety of physiological insults that are often fatal to fish and other aquatic organisms. The major symptoms that characterize Gas Bubble Trauma in fish include: 1. ) Bubble formation in the cardiovascular system. 2. ) Overinflation of the swim bladder, intestinal and peritoneal cavities. 3. ) Sub-dermal emphysema on body surfaces including the lining of the mouth. 4. ) Extracorporeal bubble formation in gill lamella. 5. ) Emphysema in muscle, internal organs and the spinal cord. In general, these symptoms involve the growth of gas bubbles in one form or another, both internal and external to the animal. Internally, bubbles can block the flow of blood, disrupt the function of organs and impair neural activity (Weitkamp and Katz, 1980; Stroud et al., 1975). Externally, they can block the flow of respiratory water through the gills (Jensen, 1980 and Shirahata, 1966). 1.1 DISSOLVED GAS SUPERSATURATION Natural bodies of water contain dissolved atmospheric gases that are usually in equilibrium with the atmosphere. That is, the partial pressures of the dissolved gas components (oxygen, nitrogen, water vapor, argon, etc.) are the same as the partial pressures of their respective atmospheric components. Occasionally, due to man made and natural causes, the dissolved gases are thrown into a state of disequilibrium with respect to atmospheric gases. When this disequilibrium involves a 2 dissolved gas partial pressure which exceeds that of the atmospheric component, water is supersaturated with that dissolved gas. This in itself may not create a problem for aquatic organisms. However, a unique condition arises when the sum of the partial pressures of all dissolved gases, (i.e. total dissolved gas pressure, henceforth referred to as Total Gas Pressure or TGP) exceeds atmospheric pressure. When this occurs, there is the potential for dissolved gases to diffuse into microscopic nucleation sites or hollow cavities and form bubbles (Harvey, 1951; Hills, 1977; Weitkamp and Katz, 1980; Fidler, 1985). These bubbles can form in both the environmental water and within the organisms that inhabit that water. Expressed in a mathematical form, the necessary condition for bubble growth and overinflation of body cavities is: TGP = p 0 2 + p N 2 + pH 2 0 + pEtc. > PAtms. where p 0 2 = the partial pressure of dissolved oxygen. p N 2 = the partial pressure of dissolved nitrogen. p H 2 0 = the partial pressure of water vapor. pEtc. = the partial pressure of all other dissolved gases. pAtms. = atmospheric pressure. In general, the partial pressures of argon and other trace atmospheric gases are small in relation to those of oxygen, nitrogen and water vapor. Thus, most studies of Gas Bubble Trauma, include trace gases in the partial pressure of nitrogen (Colt, 1983 and 1984). Occasionally, high levels of dissolved C 0 2 occur in the environmental waters. This usually involves high densities of fish in poorly aerated water such as 3 occurs in certain aquaculture operations (Steffensen, 1988 and Rosenthal, 1988: personal communications). In these situations, CO2 must be included with the other major gas components as part of the Total Gas Pressure. Equation 1.0 defines the necessary condition for bubble growth. However, this is not a sufficient condition. Physical constraints such as the surface tension of the medium in which the bubbles grow, water depth, atmospheric pressure and water temperature coupled with a variety of physiological constraints further restrict the conditions under which bubble growth and body cavity overinflation can occur (Fidler, 1985). The relationship between these parameters and bubble growth thresholds is the central theme of this thesis. 1.2 CAUSES OF SUPERSATURATION Supersaturation of natural bodies of water occurs as a result of both man made and natural phenomena. Perhaps the most widely known form of supersaturation is that produced by hydroelectric dams. Water, spilling over dams, entrains air as it plunges into pools at the base of the dam. The air, in the form of bubbles, is forced into solution under hydrostatic pressure and increases water dissolved gas tensions. Coutant and Genoway (1968), Beiningen and Ebel (1970), Bouck et al. (1970), Boyer (1974), Dell (1975), Meekin and Turner (1974), Dawley et al. (1976), Ebel (1969, 1971 & 1979), Ebel et al. (1971), Ebel et al. (1973), Ebel et al. (1975), Ebel et al. (1979), Blahm et al. (1973), Blahm (1975), Stroud and Nebeker (1976) and Weitkamp (1974 & 1976) report the effects of this form of supersaturation on fish in the Columbia River system of the United States. Supersaturation resulting from other dams and hydroelectric installations are described by Colt (1984), Berg et al. (1984), White et al. (1986), Heggberget (1984) and Alderdice and Jensen (1985). 4 DeMont and Miller (1972), Becker (1973), Adair and Hains (1974), Miller (1974) Marcello and Fairbanks (1976), and Fairbanks and Lawton (1977), report that thermal waste water from steam or nuclear power generation can raise the temperature of receiving waters, reduce dissolved gas solubility, and produce supersaturation. Natural supersaturation occurs in both the marine and fresh water environment. Harvey (1967) describes a fresh water lake in which solar radiation increased water temperatures to produce a Total Gas Pressure of 1.1 to 1.2 Atms. Similarly, Reintjes (1969), Westman and Nigrelli (1955), and Zaitsev (1971) report supersaturation caused by solar heating in ocean environments. Well water, commonly used in aquaculture, is often highly supersaturated with dissolve nitrogen (Marsh, 1910; Rucker and Tuttle, 1948; Matsue et al., 1953). Jarnefelt (1948), Ebeling (1954), Holl (1955), Lindroth (1957), and Harvey and Cooper (1962) report high levels of dissolved gases below water falls and certain types of rapids. A combination of solar heating and phytoplankton blooms can also produce high levels of supersaturation. Woodbury (1941), Alikunhi et al. (1951), Schmassmann (1951), Rukavina and Varenika (1956), Renfro (1963), and Supplee and Lightner (1976) describe many of these occurrences. Renfro (1963) reports of massive fish mortality in Galveston Bay as a result of solar heating and oxygen production by phytoplankton. Dissolved oxygen concentrations of 250% of equilibrium were recorded in this incident. White et al. (1986) describe excessive total gas pressures produced by a combination of a dam and algae blooms on an inland river. Water can also become supersaturated as a result of reducing ambient pressure. Hauck (1986) reports of mortalities incurred in pink salmon as a result of moving fry 5 by helicopter. A reduction in ambient pressure caused by a rapid increase in altitude quickly reduced gas solubility, thereby supersaturating the water. 1.3 SYMPTOMS OF GAS BUBBLE TRAUMA The symptoms of GBT are surprisingly varied. The major ones are outlined in the initial introduction to this section. In virtually all cases, bubbles are responsible for the observed symptoms. The activation of symptoms, however, may not be an easily demonstrated cause and effect relationship. This is because internally bubbles can grow in all body compartments and produce disruptions of neurological, cardiovascular, respiratory, osmoregulatory and other physiological functions. Clearly, there are opportunities for both direct and indirect effects of bubble growth. GBT can also involve a combination of bubble induced physiological stress and bacterial, viral and fungal infections (Weitkamp, 1976; Nebeker et al., 1976 and Meekin and Turner, 1974). The definitive, although slightly dated, review of GBT in fish is that of Weitkamp and Katz (1980). Their review examines almost 200 papers on the subject and describes in great detail the many symptoms of GBT. In most cases, the conditions that produce the symptoms are also specify. The more recent review by Colt, Bouck and Fidler (1986) adds further information to the overall understanding of GBT, its symptoms, causes and treatment. 1.4 REPORTING OF DISSOLVED GAS TENSIONS Early in the study of dissolved gas supersaturation, it was commonly believed that the symptoms of GBT and mortality were independent of water dissolved oxygen concentrations. As a result, many data in the literature are reported as a function of 6 p N 2 only. However, Fidler (1985), Dawley and Ebel (1975) and Rucker (1975) give evidence that the Total Gas Pressure as well as water p 0 2 levels control bubble growth and time to mortality. Thus, data from the literature based only on dissolved nitrogen measurements are of little use in obtaining correlations between water dissolved gas levels, symptoms, time to mortality or bubble growth thresholds. Because of the inconsistent manner in which many data on GBT and gas supersaturation are reported in the literature, Colt (1983 and 1984) derived the various equations for the reporting of Total Gas Pressure and other dissolved gas tensions. In more recent studies of GBT and supersaturation the standards proposed by Colt have been followed. 1.5 EXPERIMENTAL STUDIES GBT research reported in the scientific literature focuses on the identification of symptoms and time to mortality as the primary response of fish to supersaturation (Blahm et al., 1973; Blahm et al., 1975; Dawley and Ebel, 1975; Dawley ef al., 1975; Knittel et al., 1980; Meekin and Turner, 1974; Nebeker and Brett, 1976; Nebeker et al., 1979a and 1979b; Nebeker et al., 1978; Rucker, 1974; Jensen et al., 1985; Stroud et al., 1976; and Weitkamp, 1976). With few exceptions, there is no attempt to clearly identify the causes of mortality or thresholds that may be associated with mortality. Stroud and Nebeker (1976), Stroud ef al. (1975) and Meekin and Turner (1974) provide a correlation of various symptoms of GBT with water TGP levels and time to mortality. Knittel et al. (1980), in studies of Steelhead trout, offer the only description of a threshold for mortality. 7 Other studies of GBT use decompression as a means of simulating supersaturation. Beyer (1976a and 1976b), Casillas et al. (1975), Casillas (1976a and 1976b), D'Aoust and Smith (1974) and Feathers and Knable (1983) show that upon decompression, fish exhibit internal symptoms similar to those seen in fish exposed to gas supersaturated water. That is, bubble formation is prevalent in virtually all body compartments. Unfortunately, there are no distinctions made of important differences between decompression and supersaturation in fish. For example, there is little attention given to the direction of dissolved gas movement. In dissolved gas supersaturation this movement is opposite to that in decompression. That is, during decompression, dissolved gases move from body compartments into the environmental respiratory medium. However, in supersaturation, dissolved gases move from the environmental respiratory medium into body compartments. Nor is the distinction made that, during decompression, respiration reduces dissolved gas tensions in the animal; whereas, in supersaturation, respiration increases dissolved gas tensions. Perhaps the most important point overlooked is that bubble growth during decompression must take place within minutes or hours. Otherwise, dissolved gas tensions decrease as a result of respiration to levels that prevent bubble growth (Hills, 1977). In many situations involving supersaturation, bubble growth has no time limit. Fish may spend months or their entire lifes in supersaturated water (Weitkamp and Katz, 1980 and White et al., 1986). In the literature, there is little reported in the way of physiological measurements for fish exposed to supersaturation. Casillas et al. (1975, 1976a and 1976b) describe changes in an array of blood parameters with particular emphasis on clotting mechanisms in fish undergoing decompression. Newcomb (1976) describes the changes in blood chemistry in Steelhead trout exposed to supersaturated water. For 8 the most part, histological studies have involved visual examination for bubbles in various body organs and external skin surfaces. Recently, Smith (1988) has obtained photomicrographs that show the presence of micro-nuclei and bubbles in heart and gill tissue. A large volume of data were added to the literature as a result of many studies conducted on the Columbia River system in the United States. From this work, it is clear there is little in the way of a detailed understanding of the relationship between the physical causes of GBT and the physiological effects. For example, it is not known which symptoms lead to observed mortalities. Bubble growth in the vascular system is often cited as a cause (Weitkamp and Katz, 1980). Yet, other symptoms, such as extracorporeal bubbles in the gills and sub-dermal bubbles on the skin and in the mouth, are often present at the same time (Stroud and Nebeker, 1976; Stroud et al., 1975 and Meekin and Turner, 1974). It is not clear whether the various symptoms act in concert or if different lethal symptoms are separated by water TGP, p 0 2 levels or other parameters. Furthermore, it is surprising there is no information on the overall cardiovascular or respiratory response of fish exposed to supersaturated water. It is often speculated that death is due to anoxia caused by the growth of intravascular bubbles (Stroud et al., 1976; Bouck, 1980 and Newcomb, 1976). Yet, there are no measurements of arterial blood oxygen tensions under conditions of supersaturation. Other cardiovascular parameters (blood pH, blood pressure, heart rate, hematocrit) and respiratory parameters (ventilation frequency and volume) which would lead to an improved understanding of the response, are also absent from the literature. 9 Finally, there has been no attempt to determine if data from the literature exhibit differences in response to supersaturation in terms of time to mortality. That is, it is not known if mortality data show thresholds associated with specific levels of water TGP, p02, fish species or fish size. 1.6 ANALYTICAL MODELS 1.6.1 TIME TO 50% MORTALITY: The only significant attempt to relate mortality data to water parameters, species and fish size is that of Jensen, Schnute and Alderdice (1986a and 1986b). In this work, data records from the literature that include water TGP, p02, time to 50% mortality and other parameters (Jensen et al., 1985), were incorporated into a generalized surface response analysis (Schnute and McKinnell, 1984). The various models produced by this analytical technique reflect the detail to which specific physical and physiological parameters are included in the models. In this work the authors assume that there is a single cause of mortality in fish exposed to supersaturated water. That is, the data used in the various models were not examined to determine if multiple thresholds for mortality were present. If more than one threshold is present, it is reasonable to suspect that mortality is caused by more than one factor. Furthermore, these factors may be separated by water TGP, PO2, fish size, species, etc. This problem will be examined more fully in Sections 3 and 4 of this thesis. 1.62 BUBBLE GROWTH THRESHOLDS: In an earlier development by this author, equations were derived that describe thresholds for various forms of bubble growth in fish exposed to supersaturated water. The derivation involves a mass balance applied to the movement of dissolved gases from the environmental water, across the 10 gill membrane and into the blood and nucleation sites in the cardiovascular system. The resulting equations contain physical parameters related to the environmental water that include TGP, pC>2, temperature and depth. Other physical parameters in the equations account for barometric pressure, the solubilities and diffusivities of oxygen and nitrogen in water and blood, the vapor pressure of water, the surface tensions of water and fish blood and the mass transfer coefficients for the movement of dissolved gases into a growing bubble. The physiological parameters include the ratio of the partial pressure of oxygen in blood to the partial pressure of oxygen in the environmental water, the system pressure where bubble growth occurs and the size of nucleation sites from which bubble growth begins. The derivation of these equations is described in Appendix A of this thesis. Fidler (1985) gives a discussion of the role gaseous nucleation sites play in the growth of bubbles. As described in Appendix A, if the size of a nucleation site is taken as very large, such that surface tension forces are small, the threshold equation can be applied to the problem of swimbladder overinflation in fish. Yet, a further simplification of the equation yields a description of thresholds for bubble growth in the environmental water. The final results are three equations which describe the thresholds for bubble growth in the vascular system, the swimbladder and in the environmental water. Table I gives definitions of the terms appearing in the equations. From Appendix A, the threshold equations are: 11 THRESHOLD CRITERIA FOR BUBBLE GROWTH IN THE VASCULAR SYSTEM 2 a T G P C V ^ PAtm + P s + e*9-h + p02-(K-F - 1) - (1 - K)-p 0b r o Equation 1 where the subscript CV refers to the vascular system. THRESHOLD CRITERIA FOR OVERINFLATION OF THE SWIMBLADDER T G P S B * pAtm + P s + 0-g-h-p0 2-(K-F- 1)-(1 - K ) - p 0 b Equation 2 where the subscript SB refers to the swimbladder. THRESHOLD CRITERIA FOR BUBBLE GROWTH IN ENVIRONMENTAL WATER 2 a T G P E W > P A t m + Ps + p-g-h + + (K - 1)-(p 0 b - P0 2 ) r o Equation 3 where the subscript EW refers to Environmental Water. 12 TABLE I NOMENCLATURE TGP = p 0 2 + p N 2 + pH 2 0 p 0 2 = partial pressure of dissolved oxygen in environmental water. p N 2 = partial pressure of dissolved nitrogen in environmental water. pH 2 0 = vapor pressure of water. POb = P a r t ' a ' pressure of oxygen in nucleation site. r 0 = the radius of the critical nucleation sites from which bubble growth begins (environmental water or vascular system), K = H 0 - k 0 / H N - k N where, H Q = Henrys constant for oxygen in fish blood, Hjxj = Henrys constant for nitrogen in fish blood, kg = mass transfer coefficient for oxygen into nucleus, kJSJ = mass transfer coefficient for nitrogen into nucleus, T = temperature in degrees C . o = surface tension of water or fish blood. F = oxygen uptake ratio across gills, = p 0 2 (Arterial Blood)/p0 2 (Water), P = Patm. + p-h + Ps, Patm = atmospheric pressure, Ps = system pressure where bubble is growing , p = density of water, h = depth of fish in water column, g = gravitational constant. 13 Most of the physical parameters contained in the equations are well defined. However, when the equations were first derived, there was no information regarding the surface tension of fish blood. Later, Fidler (1985) found it to be essentially the same as water. In situations involving substantial depth, where fish are free to roam within that depth, it may be difficult to specify the depth term in the equations. However, in environments, where fish are confined to specific depths, all physical parameters contained in the equations are known or can be measured directly. The situation involving the physiological parameters is not as easily resolved. Although there is information in the literature that may allow the calculation of the gill oxygen uptake ratio (F) the remaining parameters, Ps and r 0 are without definition. Thus, it is not possible to define thresholds or determine if the thresholds are distinctly different in terms of water TGP and pG^. It is known that these forms of bubble growth are present in fish that have died as a result of exposure to supersaturated water. However, it is not known whether there are thresholds for mortality that may be directly correlated with thresholds for bubble growth. 2.0 STUDY DEFINITION Based on the potential usefulness of the threshold equations, an initial goal of this study was to determine the unknown physiological parameters needed to complete the equations. Next, it was necessary to establish that these forms of bubble growth are in fact responsible for physiological stress and mortality. Without this correlation, the threshold equations are of limited value and of little practical use. Thus, a vital part of the study involved an examination of the physiological response of fish to supersaturation and bubble growth. This response was of fundamental importance in achieving a correlation between mortality and bubble growth thresholds. The work was performed in three phases as outlined below. 14 Phase 1.) A theoretical analysis expanded the threshold equations and added to their usefulness in relating mathematical parameters to measurable physiological parameters. Additional mathematical models were introduced that describe the bubble growth process more fully. Phase 2.) GBT data from the literature were reviewed. A compilation of those data into a database aided in correlating observed thresholds for mortality to thresholds for bubble growth. Phase 3.) An experimental program provided further definition of the physiological parameters contained in the threshold equations. The experimental data also allowed physiological symptoms to be related to bubble growth thresholds. The results of the three phases of study were finally combined into a correlation of the bubble growth threshold equations with experimental data from this work and from the literature. 3.0 THEORETICAL STUDIES 15 3.1 INTRODUCTION As outlined above, the purpose of this study was to extend and verify the GBT bubble growth threshold models described in Appendix A. In proceeding toward this goal, it became clear that various facets of the problem were amenable to additional mathematical analysis. First, it was necessary to modify the bubble growth threshold equations. The modifications improved their utility and allowed direct methods for evaluating physiological parameters. It was also useful to expand the equations to apply to thresholds for sub-dermal bubbles. As described in the introduction, these bubbles appear on external skin surfaces and in the lining of the buccal cavity. The importance of this threshold became apparent in the experimental studies described in Section 5 of this thesis. In addition, mathematical models were developed that provide approximate descriptions of bubble growth and the interaction of growing bubbles with cardiovascular system pressure. The solutions to these equations allowed an assessment of the time course for bubble growth and indicated experimental methods for determining the effective dimensions of vascular system nucleation sites. This section will begin with an examination of the threshold equations and the parameters contained in the equations. 3.2 THRESHOLDS FOR BUBBLE GROWTH The bubble growth threshold equations were derived in the forms shown in Equations 1, 2 and 3. In general, the equations imply that thresholds for bubble growth increase with increasing water depth (h), system pressure (Ps), and barometric pressure ( P A t m s ) . On the other hand, thresholds decrease as nucleation site radius (r0), increases. The effect of water p02 on bubble growth thresholds is dependent on the 16 relative magnitude of the transport parameters (i.e. mass transfer coefficients and Henrys constants). In Equations 1 and 2, the effect of water p02 is also dependent on the oxygen uptake ratio (F) across the gill. In general, as F increases, the effect of p 0 2 on threshold TGP diminishes. Finally, the partial pressure of oxygen in the initial nucleus plays a role in the TGP thresholds. The importance of bubble oxygen partial pressure is examined in more detail later in this section. Physical parameters contained in the equations (water temperature, depth, p 0 2 and transport parameters along with surface tension) are either definable, measurable or are controlled for many situations in which the equations can be applied. For example, mass transfer coefficients, diffusion coefficients and Henrys constant for most atmospheric gases in water and fish blood are known as a function of temperature (Epstein and Plesset, 1950; Plesset, 1964; Gift et al., 1978; Altman and Dittmer, 1961, 1964 & 1971; Weiss, 1970 and Boutilier ef al., 1984). Also, the surface tension and vapor pressure of water are defined as a function of temperature (Perry, 1983 and Reid et al., 1977). The surface tension of Rainbow trout blood, at a temperature of 7° C , was determined by Fidler (1985), and found to be close to that of water. Thus, the principal unknown parameters are physiological. These consist of the ratio of arterial p 0 2 to water p 0 2 , the vascular system pressure where initial bubble growth begins and the size of critical nucleation sites. In order to examine these parameters in more detail, each threshold equation will be considered separately. 3.2.1 VASCULAR SYSTEM BUBBLE GROWTH: As described in the introduction, this symptom is commonly seen in fish exposed to dissolved gas supersaturation. It is considered to be the most lethal of all GBT symptoms (Stroud et al., 1976; and 17 Bouck, 1980). Although not stated explicitly in the threshold equation, there is a strong coupling of the nucleation site location with system pressure and oxygen uptake ratio. The system pressure (Ps) and oxygen uptake ratio (F) are specifically those at the location of the nucleation site. Fidler (1985) has pointed out that the most likely location for initial intravascular bubble formation is the arterial side of the circulatory system. This is because venous levels of dissolved oxygen are on the order of 20 - 30 mmHg.; whereas, arterial values are 100 - 130 mmHg (Holeton and Randall, 1967; Randall, 1970; Davis and Cameron, 1971; Sovio et al., 1981; Thomas and Hughes, 1982; Wood and Jackson, 1980 and Wood et al., 1984). Because of the lower venous p02, the effective TGP for venous blood is significantly lower than that of arterial blood. This indicates that much higher levels of water TGP are required before the thresholds for bubble growth in venous blood are reached. Thus, the F term in Equation 1 applies primarily to arterial blood. In general, the location of nucleation sites in the vascular system is unknown. Therefore, it is not possible to define Ps and F directly. Furthermore, the size of nucleation sites in physiological systems are unknown and this leaves the r 0 term in the equation without definition. As pointed out earlier, the threshold for intravascular bubble growth is dependent on water p 0 2 . This dependency is a result of differences between blood and water p 0 2 caused by oxygen transport resistances at the gill (Randall, 1970; Randall, 1984 and Piiper and Scheid, 1984). The ratio of blood p 0 2 to water p 0 2 has not been determined explicitly. However, there may be sufficient data in the literature to make reasonable estimates of this ratio at the dorsal aorta. With F defined at the dorsal 18 aorta, there remains the problem of determining an appropriate value of F at nucleation sites. 3.2.2 SWIMBLADDER OVERINFLATION: The swimbladders of most fish are highly vascularized with arterial blood (Steen, 1970). Thus, there is the potential for dissolved gases to overinflate this organ (Fidler, 1985; Shrimpton et al., 1988). Overinflation of the swimbladder and other body cavities accompanied by occasional mortality are reported by Shirahata, (1966); Krai, (1983); Bowser, (1983); Cornacchia and Colt, (1984); Johnson and Katavic, (1984); Kolbeinshavn and Wallace, (1985); Jensen, (1987) and Shrimpton et al., (1988). Equation 2 was derived as a special case of Equation 1. As described in Appendix A, the swim bladder acts as an extremely large nucleation site. As such, surface tension effects are small which permits the 2o/r 0 term to be dropped from the general threshold equation. Equation 1 reduces to Equation 2 where the only physiological unknown is F, the ratio of arterial p 0 2 to water p 0 2 . As pointed out above, there may be enough data in the literature to define this term for arterial blood at the dorsal aorta. However, it is still not known how the value of F at the swimbladder is related to F at the dorsal aorta. 3.2.3 WATER BUBBLES: Equation 3 is again a special case of Equation 1 where, for bubble growth in the environmental water, F is equal to 1.0. This reduces Equation 1 to Equation 3 where the only physiological unknown is the size of nuclei from which bubble growth begins. Again, this dimension is virtually unknown for physiological systems. Situations in which the equation can be applied include extracorporeal bubble growth in the buccal cavity or extracorporeal bubble growth between gill lamella. In the first case, it is observed that a single bubble in the buccal cavity of 19 larval fish can block respiratory water flow and cause death (Shirahata, 1966 and Jensen, 1980). Extracorporeal bubbles growing between gill lamella are observed in fish exposed to gas supersaturated water (Weitkamp and Katz, 1980). However, it has not been established that these bubbles are lethal to fish. 3.3 BACKGROUND AND METHODS 3.3.1 EQUATION MODIFICATION: The threshold equations can be simplified through an assumption regarding the initial partial pressure of oxygen in the nucleus. Before specifying this assumption, recall that the threshold equations are dependent on the partial pressure of oxygen in the nucleation sites (Pob)- lf> a t t i m e z e r o i n t n e bubble growth process, this pressure is out of equilibrium with the dissolved gases in solution, there are a wide range of situations where oxygen and nitrogen could be undergoing countercurrent or cocurrent diffusion. For example, it is possible for bubble growth to occur when oxygen is diffusing outward from the nucleus while nitrogen is diffusing inward. Growth of the nucleus would imply that the net outward movement of oxygen is more than offset by the inward movement of nitrogen. The opposite situation can also occur. There are, of course, those situations where all gases are diffusing into the bubble. Thus, the equations involve the coupling of transport terms to initial conditions of bubble and water oxygen partial pressures. This accounts for the many possible directions and relative magnitudes of gas transport. The effect of transport parameters on nitrogen is implicit in the equations through the definition of Total Gas Pressure (Table I). Upon exposure to supersaturated water, the development of supersaturation within fish occurs gradually. That is, at time zero in the bubble growth process, gases in 20 nucleation sites are in equilibrium with those in the surrounding medium. The subsequent disequilibrium is not instantaneous, but develops over a finite period. Thus, it will be assumed that all gases in nucleation sites are in equilibrium with the dissolved gases in the surrounding medium (i.e. water or plasma) before bubble growth begins. This allows P Q d ' n Equations 1 and 2 to be replaced with F»p02. With the same assumptions regarding bubble growth in the environmental water, P Q D is replaced with p02 in Equation 3. These substitutions allow the threshold equations to be written in the simpler forms given by Equations 4, 5 and 6. Again, Table I defines the terms appearing in the equations. THRESHOLD CRITERIA FOR BUBBLE GROWTH IN THE VASCULAR SYSTEM 2 a T G P C V > P A t m + Ps + p-g-h + + p02-(1 - F) r o Equation 4 THRESHOLD CRITERIA FOR OVERINFLATION OF THE SWIMBLADDER T G P S B > P A t m + Ps + p-g-h + p02-(1 - F) Equation 5 THRESHOLD CRITERIA FOR BUBBLE GROWTH IN ENVIRONMENTAL WATER 2 a T G P E W > P A t m + Ps + p-g-h + r o Equation 6 21 It will be noted that transport terms are absent in the new forms of the equations. The assumption of initial equilibrium between gas phases (i.e. in the bubble and in solution) assures that any increase in TGP above the threshold level will result in bubble growth. It is also noted that only Equations 4 and 5 retain a dependency on water p02. Again, this is because plasma p02 is reduced from that of the water by the factor F. 3.3.2 GILL OXYGEN UPTAKE RATIO: Throughout the literature there have been many reported measurements of arterial blood p 0 2 from the dorsal aorta of fish. In most cases water p 0 2 is also reported. In order to use this information for determining appropriate values of F, it was tabulated for Rainbow trout as shown in Appendix B. Included in the tabulation are data from the experimental phases of this investigation (Section 5). Table I of Appendix B defines the abbreviations used in the tabulation while Table II of the appendix identifies the data sources. Only data obtained for resting fish were selected for this tabulation. This was done in order to limit the variation in the data and to yield results that were applicable to the experimental phases of this study. The data were converted to F values and a mean along with standard deviations were calculated. This was done for data in the p 0 2 range of 70 to 350 mmHg only. The selection of this range is explained more fully in the discussions that follow. 3.3.3 ARTERIAL BLOOD PRESSURE: The blood pressure in vertebrates varies considerably throughout the circulatory system. In general, the pressure at any point in the system is a function of both the local fluid velocity and all friction pressure losses occurring upstream of that point (Folkow and Neil, 1971 and Welty et al., 1976). In fish, blood pressure is highest in the ventricle of the heart and decreases 22 continuously as blood moves along the arterial system toward tissue capillary beds (Randall, 1967a; Randall, 1983; and Kiceniuk and Jones, 1977). It continues to decrease in the venous system and reaches a minimum in the sinus venosus just upstream of the heart. Holeton and Randall (1967a) measured values of 60 to 70 mmHg. for blood pressure in the ventral aorta of Rainbow trout. Blood pressure in the dorsal aorta of the same species ranges from 20 to 50 mmHg. (Holeton and Randall, 1967a. also see Section 5 of this thesis). In Rainbow trout, the gills account for nearly 20% to 40% of the pressure loss between the ventral aorta and the venous return circulation (Holeton and Randall, 1967a; Stevens and Randall, 1967a). The next major drop in systemic pressure occurs in the arterioles just upstream of capillary beds (Feigl,1974). Although pressures have been measured at many locations in Rainbow trout vascular systems, there are no data for the arteriole or capillary levels. However, blood pressures have been measured on the venous side of the circulatory system of Rainbow trout by Kiceniuk and Jones (1977). These pressures are on the order of 5 to 7 mmHg. Based on these measurements for the dorsal aorta and venous system, capillary pressures are estimated to be on the order of 10 to 15 mmHg. or less (Farrell, 1988: personal communication). Swimbladder Overinflation: The swimbladders of physostome fish, are highly vascularized with small arteries and capillaries (Fange, 1966; Steen, 1970; and Steen and Sund, 1977). In Equation 5, which applies to overinflation of the swimbladder, Ps should correspond to the pressure at the swimbladder capillaries. As with tissue arterioles and capillaries, there are no blood pressure measurements available for these regions in fish. However, as suggested above, these pressures should be low and perhaps on the order of 10 to 15 mmHg. 23 Vascular System Bubbles: For vascular system bubbles, Ps must decrease as Equation 4 is applied to arterial regions more and more distant from the heart. As a result, the thresholds for vascular system bubble growth also decline. However, there is a limit to this decline; for at the capillary beds, arterial p 0 2 decreases as oxygen from the blood diffuses toward cells. This lowers the local TGP of the blood which raises the water TGP thresholds required to initiate bubble growth. This is equivalent to decreasing the F term in Equation 4 for locations in the capillary beds and beyond. Based on this analysis, it appears that the most likely sites for bubble growth are the arterioles just upstream of the tissue capillary beds. Here system pressure is at its lowest before the decline in p 0 2 begins at the capillaries. Although measurements of pressure at arteriole and capillary locations would be helpful, they are of limited value without confirmation of nucleation and bubble growth at these sites. 3.3.4 CRITICAL NUCLEI RADIUS: The r 0 term in Equations 4 and 6 is the critical radius of nucleation sites needed to start bubble growth for specific levels of water TGP and p 0 2 . In studies of decompression in humans and animals, there have been many attempts to define the size of corporeal nuclei (Yount, 1979; Yount and Yeung, 1979; Yount, 1981; Philp et al., 1972; Ackles, 1973 and Hemmingsen, 1986). Unfortunately, little quantitative information is available regarding their size. However, it is clear that size may vary with individual and with the number and frequency of previous decompression episodes (Hills, 1977). Using rats, Philp, Inwood and Warren (1972) have demonstrated that following decompression involving bubble formation, nuclei are free in the blood and are significantly larger than the original nuclei from which the bubbles formed. The residual nuclei appear to be bubbles that 24 have stabilized during collapse through the accumulation of blood protein components at the bubble surface (Philp, Inwood and Warren, 1972). This is an important finding from the standpoint of multiple decompression episodes. In fish, exposed to supersaturation, these larger nuclei would require lower TGP thresholds to initiate bubble growth during subsequent exposures to supersaturation. Knittel et al. (1980) have shown this effect in Steelhead trout. This author (1985), pointed out that if vascular system nuclei are free in the blood, they could be no larger than erythrocytes. As it is, erythrocytes are just able to squeeze through sections of gill secondary lamella and tissue capillaries (Randall, 1970; Randall and Daxboeck, 1984 and Farrell et al., 1980). In Rainbow trout, free nuclei would be on the order of 10 to 15 / L / M . in diameter; which is the characteristic dimension of erythrocytes in this species (Heming, 1984a and Mott, 1957 and Smith et al., 1952). However, there are indications that prior to the first episode of decompression, nucleation sites are not free in the blood but are associated with the walls of the vascular system. Harvey et al. (1944) could not produce bubble growth in blood from monkeys and rats, in vitro, at high levels of decompression. However, in vivo bubble growth was easily attained at comparatively low levels of decompression. Similarly, Hemmingsen et al. (1985) using in vitro studies could not produce bubbles in mammalian, avian or amphibian blood with decompression from 300 Atms. Thus, it appears that nuclei, rather than being free in blood, are in some way associated with the linings of the vascular system. This is consistent with the long held theory (Harvey, 1951) that nucleation sites are gas filled discontinuities in surfaces. Although this seems to conflict with the observations of Philp, Inwood and 25 Warren (1972), it should be recalled that their results were obtained following decompression. Thus, it is conceivable that during decompression, bubbles can grow to sizes that allow blood flow to remove them from their original sites. However, the dimensions of capillary bed vessels would be a restriction in their size and movement. It will be recalled from a previous discussion that as blood moves from the heart to the capillaries, there is a progressive decline in blood pressure. Equation 4 implies that at a given water TGP and pC>2, the size of nucleation sites required to initiate bubble growth increases as blood pressure increases. This is illustrated by rewriting Equation 4 in the following form. 2 a Ps = TGP (1-F) -p0 2 r o The solution of this equation is shown in Figure 1 where the nuclei radius required for bubble growth is plotted versus blood pressure. Other parameters used in the equation are as indicated in the figure notes. The levels of TGP and p 0 2 specified in the figure are representative of those frequently reported in the literature where mortality and vascular system bubble growth are observed. As indicated earlier, absolute values of pressure are not known at all locations in the vascular systems of fish. However, it is known that for the arterial system in Rainbow trout, the range of pressures shown in Figure 2 are representative (Stevens and Randall, 1967a,b and Kiceniuk and Jones, 1977). It is surprising that for this range, the size requirements for nuclei vary only by a factor of three. If other levels of water TGP and p 0 2 from the literature are examined, it is found that, over the range of these variables found in GBT, nuclei required for bubble growth range from 10 to 40 fjM. in radius. THRESHOLD NUCLEI RADIUS VERSUS VASCULAR SYSTEM PRESSURE TGP • 1.2 Atms. p02w • 195 mmHg. FIGURE 1 Threshold Nuclei Radius Versus Blood Pressure. 27 In situations involving bubble growth in the environmental water, nucleation sites can also be associated with the surfaces upon which bubble growth begins. This would include the external skin surfaces of the animal and the lining of the buccal cavity. It should be anticipated that extracorporeal bubble growth in the buccal cavity of small fish or in the gills of larger fish may involve nuclei carried to the site of growth by respiratory water flow. This is especially true if the environmental water carries bubbles, large silt or other particulate matter. However, nuclei radius, either associated with tissue surfaces or foreign matter, is again unknown and probably difficult to determine. Nevertheless, application of Equation 6 to a range of TGP levels typical of those found in problems of GBT, yields a radius for nucleation sites ranging from 5 to 20 yM. (Figure 2). Although the above discussion has not provided absolute information regarding the size of nucleation sites or the system pressures where bubble growth begins, it provides some insight as to the range of these parameters. There remains the problem of evaluating these parameters in an absolute sense. In intravascular bubble growth, the problem can be simplified by re-examining the threshold equations. 3.3.5 EFFECTIVE NUCLEI RADIUS: In Figure 3 the equation for vascular system bubble growth is rewritten with the Ps and r 0 terms highlighted (Equation 7). It will be noted that nucleation site radius, as contained in the 2o/r 0 portion of the equation, is a pressure term like Ps. Since both of these parameters are unknown and difficult to determine, the two can be combined into a single unknown parameter. In order to retain surface tension explicitly in the equation, the parameters are combined so that an effective radius, R 0 , accounts for both Ps and r0. This is shown as Equation 8. 28 WATER AND SKIN BUBBLE GROWTH THRESHOLDS AS A FUNCTION OF NUCLEUS RADIUS DEPTH IN METERS AS INDICATED TGP THRESHOLD - Atms. 5 6 7 8 9 1 0 11 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 NUCLEUS RADIUS - uM. Petm. - 760 mmHg. WATER TEMP. - 6 - 16 d e 0 . C. FIGURE 2: Threshold Nuclei Radius Versus Water TGP. THRESHOLD CRITERIA FOR VASCULAR SYSTEM BUBBLE GROWTH T G P > p A t m . + e-g-h + Ps + p 0 2 - ( l - F) Equation 7. 2a 2a L e t t i n g Ps + = r o R o 2a TGP > P A t m + o-g-h + + p 0 2 ( l - F) Equation 8. Ro F i g u r e 3: E f f e c t i v e Radius of N u c l e a t i o n S i t e s i n V a s c u l a r Bubble Growth Equation. 30 The result of this combination leaves Equation 8 with one unknown on the right side. Thus, if a threshold for bubble growth can be determined for known levels of water TGP and p02, or just TGP in the case of Equation 6, the R 0 and r 0 terms can be back-calculated from the equations. The equations can then be completed and used to predict thresholds of bubble growth for a wide range of conditions. Later in this section, experimental methods for determining bubble growth thresholds are examined. It should be noted that the use of an effective radius can compensate somewhat for uncertainties in the value of F at nucleation sites. That is, with F referenced to dorsal aorta blood p 0 2 , any variations between these values and those at the nucleation sites can be absorbed in the effective radius term. 3.3.6 SUB-DERMAL BUBBLE GROWTH: The literature offers many examples of sub-dermal bubbles or blisters growing on the external body surfaces of fish exposed to supersaturated water. These observations include bubbles on opercular flaps, between fin rays, and in the lining of the mouth (Weitkamp and Katz, 1980). It is hypothesized that the threshold equations apply to this form of bubble growth also. To select the appropriate equation, it is necessary to establish if dissolved gases are transported to these bubbles by blood flow through the circulatory system, or as a result of direct diffusion from the environmental water. Kirsch and Nonnote (1977) show that oxygen transport to the skin of Rainbow trout is primarily by direct diffusion from the environmental water. Furthermore, sub-dermal bubble growth appears to originate just below the epithelium tissue layer (Nebeker and Brett, 1976 and also Section 5, this thesis). Therefore, it is reasonable to assume that p 0 2 and p N 2 levels are not significantly reduced from that of water. In this case, Equation 6 would be the appropriate form of the threshold equation. 31 Aside from the dimensions of nucleation sites, the other unknown parameter is the appropriate form of surface tension. Once bubble growth actually begins, the growth rate may be controlled by a combination of extracellular fluid surface tension forces and the tensile strength of tissue (Vann and Clark, 1975; Meisel et al., 1981 and Yang and Liang, 1972). When bubbles become sufficiently large, such that surface tension forces are negligible, the tensile properties of the tissue should dominate the growth process. However, depending on the size and location of nucleation sites, the threshold situation may be controlled by water surface tension forces alone. With this assumption, Equation 6 is used as the combined water and sub-dermal bubble growth threshold equation in subsequent discussions and analyses. The validity of this assumption is examined further in Section 5 of this thesis. 3.3.7 BUBBLE GROWTH IN AN OPEN SYSTEM: It is of interest to examine the rate at which bubbles grow once threshold levels of TGP are exceeded. Time to mortality associated with GBT varies with water TGP and can range from several hundred hours at a TGP of 1.12 Atms. to just a few hours at TGP levels above 1.3 Atms. (Weitkamp and Katz. 1980). Part of this time must be associated with the period required for various body compartments of the fish to equilibrate with water dissolved gas tensions. Harvey (1963) and Beyer et al. (1976) give experimental evidence that shows this time is on the order of one to two hours. However, at high levels of TGP, bubble growth may occur before there is full equilibration between the water and all body compartments of the fish. In either case, it is expected that a significant portion of the time required to produce mortality would be that associated with bubble growth. Thus, depending on the water TGP, bubble growth periods on the order of several hundred hours to just a few hours would be expected. 32 Epstein and Plesset (1950) derive equations that describe the growth of a motionless, isothermal air bubble in still water. That is, a bubble that does not experience motion due to buoyant forces and grows as a result of diffusive gas transport. Although their derivation treated air as a single component gas (i.e. transport properties were weighted averages for oxygen and nitrogen), the resulting equations give a reasonable estimate of the time course for this type of bubble growth. For an initial nucleus radius of 10 fjM., Epstein and Plesset calculate the time for a 10 fold increase in radius to be about 550 seconds at a water TGP of 1.25 Atms. 3.3.8 BUBBLE GROWTH IN A CLOSED SYSTEM: In the derivation of Epstein and Plesset (1950), it is assumed that as bubbles grow, the volume of gas added to the system does not effect system pressure. That is, ambient hydrostatic pressure is constant. This is appropriate for bubble growth in the environmental water; such as might occur in the buccal cavity or between gill lamella. However, this is not the case for intravascular bubbles where the system is somewhat closed. In this situation, the volume of growing bubbles will eventually affect system pressure and alter the bubble growth process. The actual relationship between volume and pressure will depend on many factors; however, the compliance of the vascular system will be central to the response. The coupling of volume to pressure can be modeled mathematically in an approximate form as follows. To simplify the derivation and solution of the differential equations involved, this development will treat a single gas diffusing into bubbles forming in a closed system. The physical properties of this gas (i.e. diffusivity, Henrys constant, etc.) are taken as those of air. The derivation begins with the perfect gas relationship. n A = PA- VB/(R'-T) = (4/3)-7T-r3-PA/(R'T) 33 where, n A = moles of gas A in bubble. P A = pressure of gas in bubble. V3 = bubble volume = (4/3)-7T-r3 R' = gas constant, r = bubble radius. For ease of derivation, water vapor pressure is assumed to be small in relation to the gas pressure P A and will be neglected. Laplaces Equation relates bubble internal pressure (PA) and external pressure (PE) to surface tension and bubble radius as given by: P A = P E + (2.a/r) where, p E = pAtm. + p s + e * 9 - h pAtm. = atmospheric pressure. Ps = vascular system pressure where bubble is growing. p = density of water. g = gravitational constant. h = depth of fish in water column. For convenience, it will be assumed that the pressure in the vascular system (Ps) is composed of a system pressure Ps' existing before bubble formation and a pressure Ps" due to bubble growth. The component due to bubble growth will be a function of 34 the number of bubbles present, N, the volume of each bubble and the compliance of the vascular system, C. The component of system pressure due to bubble growth (Ps") is C times the volume of the added bubbles. That is, it is assumed that the pressure - volume relationship is linear. Assuming a uniform radius for all bubbles at any given time and that Ps' is small, Ps takes the following form. Ps = N-C-(4/3)-7T-r3 = N-C'-r3 In the expression on the far right, the constants have been lumped into a single constant, C . Substituting Laplaces Equation into the perfect gas equation, using the above definition of Ps and taking h as 0.0, gives: n A = ( 4 / 3)-7i - ( P A t m s -r3 + N C ' r 6 + 2-or2 )/(R'T) Differentiating with respect to time, t, yields: dn A 4 • 7r dt 3 R ' T dr 3 P A t m s . r Z + e-N-C'-r3 + 4 • a • r dt Equation 9. The rate of change in the number of moles of gas in a bubble must be balanced by the rate of gas added to the bubble by diffusive transport from the blood. Using the bubble mass transfer coefficient (DA/r) of Epstein and Plesset (1950), the equation for gas transfer to or from the bubble is: 35 d n 2 d t = 4 -T r-Hi r z • r D A i r P0A r - Pj Equation 10. where, H A = Henrys constant for gas A D A = Diffusivity of gas A in biood PQA = Total Gas Pressure of A in blood For this derivation, it is assumed that PQA °f 9 a s ' n t n e blood is the same as that in the water. Equating the right sides of Equations 9 and 10 yields an approximate expression for bubble growth in the vascular system of a fish exposed to supersaturated water. To arrange the equation in a form that can be integrated, variables are separated and the equation is written as: r 3 - P A t m s . - r + 6 N C ' r 4 + 4 - a - d r 3 R' T D A H A P0A " pAtms, - N C -3 _ ( 2 - a / r ) Equation 11. If the compliance term C is equal to zero, Equation 11 reduces to that for bubble growth in an open system. 36 The solution of Equation 11 is obtained numerically using the MathCad computer program. This involves substituting selected values of r into the equation and solving for t. Because the solution is asymptotic, it is possible to select values of r which are too large; in which case the numerical solution will not converge. This problem can be avoided by using Equation 11, in its differential form, and setting dr/dt equal to zero. This yields: p O A ' pAtms." N-C'-r3 - (2a /r) = 0 Equation 12. One real root of this algebraic equation gives the asymptotic value of r which is the upper limit to be used in the solution of Equation 11. The other real root is the equilibrium radius corresponding to the threshold TGP. To calculate a threshold TGP (or PQA i n t n ' s case), Equation 12 is solved for PQA- T n e result is: p O A = pAtms. + N - C ' - r 3 + (2a/r) Equation 13. Substituting r 0 for the initial nucleus radius yields the threshold TGP. 3.4 R E S U L T S 3.4.1 THRESHOLD EQUATIONS: Equations 4 through 6 are plotted in Figure 4. The plots are based on an effective nuclei radius of 10 /uM., a water depth of 0.0 M., a water temperature of 12° C , an F of 0.79 (see below) and sea level atmospheric pressure. The selection of R 0 may appear somewhat arbitrary; GBT TOTAL GAS PRESSURE THRESHOLDS 3 7 AS A FUNCTION OF WATER p02 FOR RAINBOW TROUT TGP THRESHOLD - Atms. 50 100 150 200 250 WATER P 0 2 - mmHg. 300 350 VASCULAR SYSTEM WATER AND SKIN SWIMBLADDER FIGURE 4: Theoretical Water Total Gas pressure Thresholds. 38 however, as will be shown in later sections, this value is representative. In the figure, it is seen that there is a clear separation of the various theoretical thresholds. The dependency of the thresholds for swimbladder overinflation and intravascular bubble growth on water p 0 2 is also clearly shown. Interestingly, Equations 4 and 5 now contain water p 0 2 as the only independent dissolved gas parameter. The other physical and physiological parameters are as in the original equations. Thus, the specification of water p 0 2 and temperature is sufficient to define the TGP thresholds for swim bladder overinflation and intravascular bubble growth. Although this may appear odd at first, it should be remembered that TGP is made up of the vapor pressure of water plus the partial pressures of dissolved oxygen and dissolved nitrogen as defined in Table I. By specifying water p 0 2 , water TGP is defined by the threshold equation, from which pN2 is determine by: p N 2 = TGP - p 0 2 - pH 2 0 Equation 14 With the specification of temperature, the vapor pressure of water is determined, and in turn pN 2 . Thus, the definition of p N 2 is implicit in Equations 4 and 5. 3.4.2 OXYGEN UPTAKE RATIO: Figure 5 shows the gill oxygen uptake ratios (F) calculated from the data of Appendix B, and plotted as a function of water p 0 2 . From the figure, it is clear that F is quite variable with the highest variability occurring in the region where water p02 levels are hypoxic for fish (Holeton and Randall, 1967a,b and Thomas and Hughes, 1982). Also, at the two extremes of water p 0 2 (less than 70 mmHg. and greater than 350 mmHg.), the ratio of arterial p 0 2 to water p 0 2 appears to decline. Between these two values F is estimated to have a mean value of 0.79 with a standard deviation of 0.045. In most cases involving fish exposed to RATIO OF ARTERIAL BLOOD TO WATER p02 39 FOR RAINBOW TROUT LITERATURE AND UNPUBLISHED DATA UPTAKE RATIO 1 0.9 0.8 0.7 0.6 0.6 0.4 0.3 0.2 0.1 I: -1 ' * j 1 1 J 0 50 100 150 200 250 300 350 400 450 500 550 600 WATER p02 - mmHg. FIGURE 5: Ratio of Arterial to Water p02 for Rainbow Trout. 40 supersaturation, water p 0 2 is usually elevated relative to atmospheric values (Weitkamp and Katz, 1980 and Colt, Bouck and Fidler, 1986). The maximum p 0 2 reported in the literature is about 390 mmHg. (Renfro, 1963). Cases of supersaturation involving low levels of p 0 2 usually involves well water. However, in these situations oxygen partial pressures less than 70 mmHg. have not been reported in the literature. Therefore, in subsequent analyses, the value of 0.79 for F will be assumed as representative of most situations involving supersaturation and GBT. It order to determine the effect of variations in F on the intravascular bubble thresholds, Equation 4 was examined for water p 0 2 values between 70 and 350 mmHg. Figure 6 shows the results of this analysis. In the figure, the mean value of 0.79 has been used in Equation 4 and plotted along with the standard deviations. Other parameters used in Equation 4 are as specified in the figure. From the three curves, it is clear that the effect of variations in F on threshold TGP is dependent on water p 0 2 . At a water p 0 2 of 200 mmHg., the variation in threshold TGP is as much as 0.015 Atms. 3.4.3 BUBBLE GROWTH IN AN OPEN SYSTEM: Using the Mathcad computer program, the equations for bubble growth in a closed system were first solved with the compliance (C) set equal to zero. As pointed out earlier, this reduces the growth equation (Equation 11) to that for an open system. The results of this solution are shown in Figure 7 for TGP levels of 1.31, 1.27 and 1.165 Atms. Other parameters used in the solution are shown in the figure. It is observed that the growth curve corresponding to a water TGP of 1.27 Atms. agrees closely to the solutions obtained by Epstein and Plesset (1950) for a water TGP of 1.25 Atms. In general, for an open ARTERIAL BUBBLE GROWTH THRESHOLDS TGP VS. WATER p02 FOR RO - 12 uM. AND 02 UPTAKE RATIO - .79 41 TGP THRESHOLD - Atms. 1.25 i 1.12 H 1 1 1 1 1 1 50 100 150 200 250 300 350 WATER P02 - mmHg. Water Depth - 0.0 M. Water Temperature " 5 - 1 5 deg. C. Atmospheric Pressure • 760 mmHg. i: Variation in Arterial Bubble Growth Thresholds Versus Oxygen Uptake Ratio. BUBBLE RADIUS VERSUS TIME FOR BUBBLE GROWTH IN AN OPEN SYSTEM 42 Bubble Radius - uM. 120 100 100 200 Time - Seconds 300 400 TGP - 1.31 Atms TGP - 1.165 Atms TGP - 1.27 Atms Initial Nuclei Radius - 12 uM. Temp • 15 deg. C , Depth • 0.0 M. FIGURE 7: Bubble Radius Versus Time for Bubble Growth in an Open System. 43 system, a 10 fold increase in bubble radius occurs in a time period of minutes. It should be noted that the solution to the growth rate equations is logarithmic in this case (see Epstein and Plesset, 1950). Therefore, the bubbles continue to grow with the radius becoming infinite. 3.4.4 BUBBLE GROWTH IN A CLOSED SYSTEM: Equation 11 was also solved for a finite compliance and two conditions of PQA- T n e results are shown in Figures 8 and 9. With the conditions of atmospheric pressure, temperature and critical nuclei shown in the figures, the threshold for bubble growth is 1.12 Atms. The time course for bubble growth at a TGP of 1.20 Atms. is shown in Figure 8, while Figure 9 shows bubble growth for a TGP of 1.316 Atms. In these solutions, the compliance of the system has been chosen somewhat arbitrarily. This is because compliances for the vascular systems of fish are unknown. The value of C used is 7 • 10"5 (mmHg./^/M3). Also, total bubble volume is calculated based on an assumption that 1000 bubbles are present in the system. The initial growth of these bubbles appears to follow that of an open system (Figure 7). However, once bubble volume increases system pressure, growth rate declines. It should be noted that, in a closed system, bubble growth does not proceed to an infinite radius. This is because the solution to equation 11 is asymptotic. That is, as bubbles grow, the threshold TGP also increases. This increase continues until system pressure forces the threshold to that corresponding to the actual radius of the growing bubble. Further bubble growth is then suppressed. By comparing Figures 8 and 9, it is seen that the maximum radius which bubbles reach, before growth is suppressed, increases as water TGP (or PQA in this case) increases.* BUBBLE GROWTH IN ARTERIAL BLOOD WITH BUBBLE VOLUME FEEDBACK AND VASCULAR SYSTEM COMPLIANCE 4 4 P Time - Seconds/10 Water TGP • 1.20 Atms.. Ro • 12 uM. Depth - 0 M., Temp. - 10 deg. C. System Pressured • 0) • 0.0 mmHg. FIGURE 8: Bubble Growth in a Closed System. 45 BUBBLE GROWTH IN ARTERIAL BLOOD WITH BUBBLE VOLUME FEEDBACK AND VASCULAR SYSTEM COMPLIANCE Time - Seconds/10 Water TQP • 1.316 Atms.. Ro • 12 uM. Depth - 0 M., Temp. • 10 deg. C. System Pressured • 0) • 0 mmHg. FIGURE 9: Bubble Growth in a Closed System. TGP = 1.316 Atms. 46 3.5 DISCUSSION 3.5.1 OXYGEN UPTAKE RATIO: Although the data of Figure 5 are quite variable, the values of F in the p 0 2 range of 70 to 350 mmHg. have a mean of about 0.79. This range of p 0 2 corresponds to that where arterial blood in Rainbow trout is fully saturated with oxygen (Beaumont, 1968 and Cameron, 1971) and hyperoxic conditions where respiratory effort is reduced (Randall and Jones, 1973 and Wood and Jackson, 1980). If, in this range of water p 0 2 , the gill water flow and blood flow are unaltered, the ratio of arterial p 0 2 to water p 0 2 should be unvarying. This is due to several factors. First, with constant blood and water flow, the mass transfer coefficients across the gill membrane are constant. Furthermore, as shown by Piper and Scheid (1984), for a gill counter current gas exchange system with constant diffusing capacity: (Pa - Pv)/(Pi - Pv) -+ const. < 1.0 where Pa = partial pressure of oxygen in arterial blood. Pv = partial pressure of oxygen in venous blood. Pi = partial pressure of oxygen in inspired water. Secondly, most oxygen carried by arterial blood is used in metabolism and most of this oxygen is transported in the bound form. Thus, for fully saturated blood, venous p 0 2 levels are low in comparison to either water or arterial levels, regardless of water values. In the limit, as Pv -*• 0, (Pa - Pv)/(Pi - Pv) -+ Pa/Pi -* const. < 1.0 Equation 15. 47 3.5.2 BUBBLE GROWTH THRESHOLD EQUATIONS: With the above definition of F, the bubble growth threshold equations (Equations 4 and 6) are resolved to forms containing an effective nuclei radius as the only unknown dependent variable. In the case of thresholds for swimbladder overinflation, Equation 5 is complete without a nuclei radius. The remaining task was to demonstrate its validity. In work by Mark Shrimpton, Dave Randall and this author (1988), thresholds for swimbladder overinflation in Rainbow trout were examined experimentally. Fish were exposed to gas supersaturated water while swimbladder pressures were monitored. The results of these experiments are shown in Figure 10. The square symbols in the figure represent those fish exhibiting an increase in swimbladder pressure. The plus symbols are for fish in which declining swimbladder pressures were observed. The region between the two clearly defines the threshold for swimbladder inflation. Also shown in the figure is the theoretical threshold as calculated from Equation 5. The theoretical curve of the figure is for an F of 0.79, a system pressure of 0.0 mmHg., a temperature of 10° C. and sea level atmospheric pressure. Although the experimental data do not densely cover the entire range of water p02, it is clear that the theoretical thresholds fall close to but slightly above the experimental thresholds. The reason for this difference is not presently known. However, if the value of F is increased from 0.79 to 0.85, the theoretical threshold corresponds closely to that indicated by the experimental data. It should be noted that this value of F is still within plus one standard deviation of the mean value of F, 0.79, indicated by the data of Figure 5. However, it is not clear why the higher value of F is appropriate for the swimbladder thresholds. Nevertheless, with this adjustment in F, Equation 5 provides an accurate description of thresholds for swimbladder overinflation. RESPONSE OF SWIMBLADDER TO GAS 4 8 SUPERSATURATED WATER: COMPARISON OF EXPERIMENTAL DATA WITH THEORY WATER TGP - Atms. 125 150 175 200 225 250 275 300 325 350 WATER P02 - mmHg. D INFLATION + DEFLATION THEORY Experimental Data: Shrimpton, Randall and Fidler (1988) Theory: Equation 5 FIGURE 10: Swimbladder Inflation Thresholds. 49 3.5.3 BUBBLE GROWTH RATE: Based on solutions to the bubble growth equations for open and closed systems, the time period for bubble growth is on the order of minutes to a few hours (Figures 7, 8 and 9). Thus, except at very high levels of water TGP, the time period for bubble growth does not appear to be a major component of the total time to mortality. However, this disparity in the time course of the two processes may relate to various assumptions involved in the derivation of the growth rate equations. First, the difference is probably greater than indicated in this analysis. This is because dissolved gases in the cardiovascular system are transported to the bubble by flowing blood. In this case, mass transport is mainly by convection which, for the same concentration difference, is many times more effective than transport by diffusion alone. Thus, initial bubble growth may be more rapid than indicated, which implies an even greater difference between bubble growth times and time to mortality. As bubbles grow, they should eventually block the arteries in which they are growing. Once arterial blockage occurs, blood flow stops and, as was assumed in the equation derivation, the transport of gases to the bubble is by diffusion. However, at this point, the bubble growth problem becomes one of axial diffusion rather than spherically symmetric diffusion. Also, the effective interfacial diffusion area is reduced by about one half. In addition, there will be a loss of oxygen from the bubble in a downstream direction as tissue metabolism reduces dissolved oxygen concentrations in that direction. These effects will slow the bubble growth process considerably and perhaps account for part of the time difference. Philp, Inwood and Warren (1972) have pointed out that bubble growth in blood may also involve an accumulation of protein components from the blood on the bubble surface. Casillas et al. (1975 and 1976) also report that clotting takes place in the blood of Chinook 50 salmon during decompression. The presence of these organic materials at the bubble surface may slow diffusion and the bubble growth process further. Thus, there are a number of factors that can explain some of the differences between bubble growth rates and the time required to produce mortality in fish. Further discussion of this subject is delayed until Section 5 where the results of the experimental work are examined. These results add further insight into bubble growth and yield information on the location of certain intravascular bubbles. Perhaps the most important aspect of the bubble growth model for a closed system is the indication that the volume of growing bubbles can interact with system compliance and increase system pressure. The increase in pressure will then arrest bubble growth. It is possible that increases in system pressure can provide a means for experimentally determining intravascular bubble growth thresholds. For example, if fish are exposed to gradually increasing levels of water TGP, while monitoring blood pressure, a persistent increase in blood pressure may signify the beginning of bubble growth and a TGP threshold. Once the threshold is experimentally defined, the effective radius of vascular system nuclei can be back calculated from Equation 4. This would then allow Equation 4 to be completed. A component of the experimental studies described in Section 5 of this thesis included this technique. An unknown aspect of this response is whether the pressure increase is large enough to be detected at bubble growth thresholds. In addition to the results shown in Figures 8 and 9, a sensitivity analysis of Equation 11 was performed for TGP levels close to bubble growth thresholds. In particular, for a threshold TGP of 1.12017 Atms. and a water TGP of 1.12039 Atms., bubble growth 51 is arrested with a system pressure increase of 3.8 mmHg. At a water TGP of 1.123 Atms., bubble growth is arrested by a 15 mmHg. increase in system pressure. Although Equation 11 is approximate and has components such as compliance that are chosen arbitrarily, it nevertheless suggests that, near threshold conditions, significant increases in system pressure may occur as a result of bubble growth. There are other facets to this response, such as the systemic control of blood pressure that will modify these results. However, a discussion of these effects will be postponed until a later section where the results of the experimental work are examined. 3.6 THEORETICAL SUMMARY In this section, equations that describe various facets of bubble growth in fish exposed to supersaturated water were examined. From this examination it is clear that thresholds exist for the inflation of the swimbladders in Rainbow trout exposed to supersaturated water. Based on experimental data, these thresholds are described adequately by Equation 5 using and oxygen uptake ratio (F) of 0.85. Theory also predicts the existence of thresholds for intravascular bubble growth, bubble growth in the environmental water and sub-dermal bubble growth in epithelium skin tissue. Intravascular bubble growth involves a coupling of bubble volume with the compliance of the vascular system. This coupling may lead to an increase in system pressure and a suppression of further bubble growth. By combining the system pressure (Ps) in Equation 4 with the radius of nuclei (r0), the threshold equation for intravascular growth contains one unknown parameter, the effective nucleation site radius, R 0 . Similarly, the equation for water/sub-dermal 52 bubble growth thresholds contains nucleation site radius as the only unknown physiological parameter. Based on the above analyses, several steps can now be taken to define the effective nuclei radius in the intravascular and water/sub-dermal bubble growth threshold equations. The first is to examine data from the literature and determine if there are thresholds in time to mortality associated with GBT. If so, it should be established that these thresholds can be correlated with bubble growth thresholds as predicted by equations 4 and 6. Thus, there is an experimental requirement to determine the relationship between the predicted bubble growth thresholds, observed bubble growth thresholds, and physiological parameters that relate the two to mortality. It should be re-emphasized that bubble growth thresholds are significant mainly from the standpoint of mortality and stress in fish. These next steps begin with an examination of GBT data from the literature. 4.0 GAS BUBBLE TRAUMA DATABASE 53 This phase of study involved a review of the literature on dissolved gas supersaturation and GBT in fish. The purpose of the review was to build a database of documented response to supersaturation that could be analyzed for threshold information. A secondary purpose of the review was to extract from the data other relationships that may exist between reported parameters. By building a database containing many different experimental records, it was anticipated that gaps in data from individual experiments would be filled by data from other experiments. With a more complete description of response, the chances of identifying relationships between parameters and mortality thresholds would be enhanced. 4.1 METHODS AND MATERIALS The literature review was restricted to five fish species. These included Chinook, Coho, and Sockeye salmon as well as Steelhead and Cutthroat trout. This restriction is based on two factors. The first is the abundance of data on these species compared to the relatively limited data on other species (Weitkamp and Katz, 1980 and Colt, Bouck and Fidler, 1986). The second is the physiological similarity of these animals to each other and to Rainbow trout which were used in the experimental phase of this work. The review included a re-examination of the data reported by Jensen et al. (1985a) as well as additional data not included in their review. In particular, Jensen and co-workers did not include in their database experimental records involving fish exposed 54 to supersaturation, but without observed mortality. Clearly, water TGP and p 0 2 conditions that either produce or fail to produce mortality will be strong indicators of GBT mortality thresholds. Hence, this information, was of particular importance in the database developed in this study. 4.1.1 DATA SOURCES: Table I of Appendix C lists the literature sources from which the GBT database was developed. Complete information on these sources is found in the bibliography of this thesis. The information included in the database is shown in Table II, Appendix C. Each experimental record is given a unique identification consisting of a two digit number corresponding to an Author Number. This is followed by a four digit decimal fraction corresponding to a particular data record of that author. This follows the system used by Jensen et al. (1985). In reviewing data from the literature, a number of recording errors were found in the database of Jensen and co-workers. These were corrected and noted. In addition, a number of data records were found to be inappropriate to the purposes of this study. For example, the data of Nebeker et al. (1979) are for fish that were exposed to supersaturation while undergoing sudden changes in temperature of up to 17° C. Because of the combined stress condition, these records were excluded from the database developed in this study. Further, the review of Jensen and co-workers includes only recorded data values. That is, there is no interpolation between data points. Many data records from the literature contain information on time to 50 % mortality. On the other hand, other records do not have time to 50% mortality but do have sufficient data at both lower and higher mortalities to allow a reasonable interpolation of the 50% value. That is, at least three and often four adjacent datum points are available to allow second or third order interpolations. The same is true for other levels of mortality. Consequently, this review contains interpolated results 55 where there are sufficient data. Due to differences in the approach to building this database, the record numbers listed in Appendix C do not always correspond to those of Jensen et al. (1985a). 4.1.2 METHODS OF ANALYSIS: The Gas Bubble Trauma database was analyzed using an IBM compatible personal computer running the Borland Reflex database program and the Lotus 123 spreadsheet program. The actual use of the programs depended on whether the analysis required numerical operations or filtering operations. For example, throughout the literature, one encounters two methods for reporting Total Gas Pressure. In some cases, the vapor pressure of water is included in the Total Gas Pressure calculation while in other cases it is not. For consistency, both from the standpoint of reporting and from the standpoint of the physics of the processes involved (Fidler, 1985), all TGP data were corrected to include the vapor pressure of water. In addition, where information permitted, oxygen to nitrogen ratios were converted to the partial pressures of oxygen and nitrogen. The Lotus 123 program was used to perform the calculations needed to obtain these quantities as well as other calculations used in the analysis. The Reflex program was used to develop filters that allow searching for mortality thresholds and relationships between reported parameters in the data. Data filtering is a means of searching the database for records that meet only certain criteria. For example, by restricting the Species Code to a value of 1, only data on Chinook salmon would be selected. Similarly, by setting Species Code equal to 1 and depth to 1.0 or less, only data on Chinook salmon exposed to supersaturation at a depth of one meter or less would be selected. 56 4.2 RESULTS From the literature sources listed in Table I, Appendix C, a database of 1013 data records was developed. These records consist of about 500 entries from the database of Jensen et al. (1985a). These records were corrected for entry errors and 148 records were eliminated. The eliminations, as mentioned earlier, were data from experiments of combined thermal stress and supersaturation stress (Nebeker et al., 1979). Over 500 new data records were added as a result of this review. The complete tabulation of data is contained in Table III of Appendix C. 4.2.1 PRELIMINARY FILTERING: To determine if data from the literature exhibit thresholds or other relationships between reported parameters, it was necessary to perform sorting and filtering operations on the database. For example, it is known that salmon and trout eggs are highly resistant to supersaturation and that mortalities occur only at very high levels of TGP (Alderdice and Jensen, 1985 and Rucker, 1975b). To prevent these data from obscuring trends that may be present in data for hatched fish, the first filter application was to restrict the records to those for hatched fish only. As described in the introduction to this thesis, many experimental records in the literature are based on the premise that dissolved nitrogen is the sole cause of Gas Bubble Trauma. As a result, Total Gas Pressures are not reported in these data. Thus, the second filtering of the database restricted the records to those that report TGP. Figure 11 summarizes the information to this level of filtering where time to mortality is plotted as a function of water TGP. In general, the figure shows there is a lower threshold for mortality near a water TGP of 1.1 Atms. As noted, negative times correspond to experiments where mortalities were not observed. As will be shown, 57 many of these negative times can be explained in terms of compensation,depth available to the fish or high levels of dissolved oxygen in the water. However, other data indicated by negative times are important indicators of threshold levels of TGP. The absolute value of the negative entries, is based on the duration of the experiment divided by -10. To convert this entry back to the duration of the experiment, multiply the negative time to mortality (time of survival) by -10. The levels of mortality that are included in the database range from a few percent to 100 percent. Because many of the reported data do not contain control experiments, the levels of mortality associated with non-supersaturated conditions are unknown. Furthermore, data on the time to mortality below 20% are quite sparse in the database. In order to establish a level of significance in the response to supersaturation, the database was further filtered to include only records associated with mortality levels between and including 20% and 70%. The 70% level was chosen because data for higher levels of mortality were again sparse and did not yield additional information beyond that within the 20% - 70% levels. A further restriction placed on the data at this point was to include only data for which fish size is known. The reason for this restriction will be discussed in the following sections. Using these filter criteria, the data are as shown in Figure 12. As indicated in the figure, the data have been separated based on fish length. In general, the data show that time to mortality for fish larger than 50 mm. is shorter than for fish smaller than 50 mm. Again, there is strong indication of a threshold for mortality at a TGP of 1.1 Atms. TIME T O M O R T A L I T Y V E R S U S W A T E R T G P FOR ALL FISH IN DATABASE BY L.E. FIDLER 2800 2600 2400 2200 2 0 0 0 -1800-1600-1400-1200-1000 -800 600 400 200 + 0 -200 TIME TO MORTALITY - Hrs. o o o 8 o o o o o o 8 " i tfo <*P o°ogpm> Q? o o o o o oax»° j i O OB OP B O O 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 WATER TGP - Atms. Note : Nega t i ve t i m e s c o r r e s p o n d to e x p e r i m e n t s w i th no m o r t a l i t y . FIGURE 11: Time to Mortality for all Fish in Database by LE. Fidler en oo TIME TO MORTALITY VERSUS WATER TGP MORTALITY RANGE = 20 - 70 % 2000 TIME TO MORTALITY - Hrs. 1800 1600 1400 1200 1000 800 600 4 0 0 -2 0 0 -0 200 o o v 8 o FISH < 50 mm. IN LENGTH * FISH > 50 mm. IN LENGTH o o + 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 WATER TGP - Atms. Note: Negative times correspond to experiments with no mortalality. FIGURE 12: Time to Mortality Versus Water TGP; Mortality Range = 20 - 70%. 60 The rendering of the database to this point serves as the basis from which all subsequent analyses were performed. In the descriptions that follow, the minimum level of information examined contains Total Gas Pressure and times to mortality between the 20% and 70% levels for hatched fish of known length. Also, the majority of these filtered data correspond to exposure to supersaturation at depths of 0.61 meters or less. In the entire database there are only 10 records for depths greater than 0.61 meters. 4.2.2 UNIQUE DATA SETS: Before considering the database further, it is important to examine six distinct data records. These records will appear repeatedly throughout the analyses that follow. Figure 13, which shows the time to mortality versus fish length, will be used to examine these data. First, the datum point shown by the solid black circle at 1182 hours in Figure 13 (Record No. 879) is unique in that the level of total gas pressure is low (1.13 Atms.), the depth is one meter and the fish are small (i.e. close to 50 mm. in length). It should be noted that this data record yields a time to 50% mortality almost two orders of magnitude greater than data at similar water TGP but slightly shallower depths. Next, five data sets (Records 850, 853, 856, 874, and 875) shown by the five triangular symbols in the figure correspond to a water TGP of 1.19 Atms. and p 0 2 levels above 271 mmHg. Again, the times to mortality for these data are considerably above those of data at similar TGP but lower water p 0 2 levels. The significance of these data will be explained in the discussion that follows. In the meantime it should be noted that, for fish greater than 50 mm. in length, these data are distinct from the rest in terms of time to mortality and certain water parameters. TIME TO MORTALITY VERSUS FISH LENGTH TGP = 1.08 - 1.50 Atms., MORTALITY RANGE = 20 - 70% 2800 2600 2400 2200 2000 1800 1600 1400 -1200 -1000 -800 600 400 + 200 0 -200 TIME TO MORTALITY - Hrs. • DEPTH = 1 M. A p02w > 271 mmHg. o o 0 o o ° ^ o o B o + 0 50 100 150 200 250 300 350 400 450 500 550 600 FISH LENGTH - mm. Note: Negative times correspond to experiments with no mortality. FIGURE 13: Time to Mortality Versus Fish Length; TGP = 1.08 - 1.5 Atms., Mortality Range = 20 -70 %. cn 62 4.2.3 EFFECT OF FISH SIZE: It is frequently reported in the literature that small fish are more resistant to supersaturation than large fish (Rucker and Kangas, 1974 and Jensen et al., 1986). To illustrate the effect of fish size on the mortality response, the time to mortality (20 - 70% ) is plotted versus fish length in Figure 13. Except for the six data sets mentioned above, it is clear that a significant difference exists between the times to mortality for fish less than 50 mm. in length and for those greater than 50 mm. When this relationship is examined in more detail, it is found that the difference exists independent of water TGP. In Figures 14, 15 and 16 the time to mortality is again plotted versus fish length with the exception that data have been separated based on TGP. Figure 14 gives these results for a TGP range of 1.08 to 1.15 Atms., Figure 15 for a TGP range of 1.15 to 1.20 Atms., and Figure 16 for a TGP range of 1.20 to 1.5 Atms. Although the effect of size is clearly evident in all three ranges, it is most pronounced in the 1.20 to 1.5 Atms. range. These results clearly indicate that 50 mm. is a critical length in time to mortality for these five species. 4.2.4 EFFECT OF FISH SPECIES: The data sets were next filtered according to species. The intent of this separation was to determine if differences in thresholds or other parametric relationships exist based on species. Fish Less Than 50 mm.: Figure 17 summarizes time to mortality as a function of TGP for fish less than 50 mm. in length. At this level of filtering, the database contains information on Steelhead trout and Chinook and Coho salmon only. From the data of the figure, it is apparent that a mortality threshold exists for all three species at a water TGP of about 1.12 Atms. For Steelhead trout, the range of time to mortality above a TGP of 1.15 Atms. differs from that below 1.15 Atms. That is, between a TGP of 1.1 TIME TO MORTALITY VS. FISH LENGTH 6 3 ALL FISH: TGP - 1.08 - 1.15 Atms. MORTALITY RANGE • 20 - 70 % 2000 1800 1600 1400 1200 1000 800 600 TIME TO MORTALITY - Hrs. 0 -200 _ • • - * • • • • — 7 : s • -I • • • : =: i • • | • • • > • 50 100 150 200 FISH LENGTH - mm. 250 * Depth • 1.0 m. Note: Negative timet correspond to experiments with no mortality FIGURE 14: Time to Mortality Versus Fish Length; TGP = 1.08 -1.15 Atms. 64 TIME TO MORTALITY VS. FISH LENGTH ALL FISH: TGP • 1.15 - 1.20 Atms. MORTALITY RANGE = 20 - 70 % TIME TO MORTALITY - Hrs. 1600 1400 1200 1000 800 600 400 200 0 -200 GG MS £ — £ § R Iii — E ! : n a . (=5 e I I u - | " B 13 E B ^ " B E E — »o ' at B E 1 1 " B | E B E B E a • I 1 4 : E E B I • B • " • 1 i i B 1 E J B I i 1 1 1 1 1 I 0 50 100 150 200 250 300 350 400 450 500 550 600 FISH LENGTH - mm. • p02w > 271 mHg. p02w « 271 mmHg. N o t e : N e g a t i v e t i m e s c o r r e s p o n d to e x p e r i m e n t s w i t h no m o r t a l i t y . FIGURE 15: Time to Mortality versus Fish Length TGP = 1.15- 1.20 Atms. TIME TO MORTALITY VS. FISH LENGTH ALL FISH: TGP • 1.20 - 1.50 Atms. MORTALITY RANGE - 20 -70 % 65 TIME TO MORTALITY - Hrs. 1400 1200 1000 800 600 400 200 --200 i • • L — ; . • • * . • 0 50 100 150 200 250 300 350 400 450 500 550 600 FISH LENGTH - mm. Note: Negative timet correspond to experiments with no mortality FIGURE 16,: Time to Mortality versus Fish Length; TGP = 1.20 -1.50 Atms. TIME TO MORTALITY VERSUS WATER TGP 6 6 FOR FISH LESS THAN 50 mm. IN LENGTH MORTALITY RANGE 20 - 70 % 2000 1800 1600 1400 1200 1000 800 600 400 200 0 TIME TO MORTALITY - Hrs. -200 + 1.05 0 0 + • • 0 0 I 1 + + 1.1 1.15 1.2 WATER TGP - Atms. 1.25 1.3 • STEELHEAD + COHO 0 CHINOOK Note: Negative times correspond to experiments with no mortality. FIGURE 17: Time to Mortality Versus Water TGP for Fish Less Than 50 mm. in Length. 67 and 1.15 Atms. time to mortality is relatively short compared to that just above 1.15 Atms. As TGP increases beyond 1.15 Atms., time to mortality declines to levels comparable to those below 1.15 Atms. The opposite response is observed in Chinook salmon where mortality levels below 1.15 Atms. are high in comparison to those above 1.15 Atms. For Coho salmon, there is no significant trend in time to mortality above or below 1.15 Atms. Chinook Salmon Greater Than 50 mm.: Shown in Figure 18 is the response of larger Chinook salmon to supersaturation as indicated by time to mortality versus water TGP. It is clear that a lower threshold for mortality exists at 1.11 Atms. TGP. From this threshold, time to mortality decreases as TGP increases to 1.18 Atms. At 1.18 Atms., time to mortality suddenly increases to levels comparable to those at 1.11 Atms. Above 1.18 Atms., time to mortality again declines with increasing TGP, similar to that between 1.1 and 1.18 Atms. Due to this similarity of response at 1.1 Atms. and 1.18 Atms., there may be another threshold at a TGP of 1.18 Atms for larger Chinook salmon. If so, it is not clear why the mechanism that is responsible for mortality between 1.1 and 1.18 Atms. suddenly becomes less effective at a TGP of 1.18 Atms. In fact, the data suggests a transition to another mechanism for mortality at 1.18 Atms. and above. Another interesting feature of the data is that at both 1.11 Atms. and 1.18 Atms. there is a minimum time to mortality of about 100 hours. Sockeye Salmon Greater Than 50 mm.: A somewhat different response is observed in Sockeye salmon. This is shown in Figure 19, where again time to mortality is plotted versus water TGP. For Sockeye salmon, there is no evidence of a mortality threshold near the 1.1 Atms. TGP level. However, this may be due to the sparseness of data in the region between 1.1 and 1.17 Atms. On the other hand, there is strong TIME TO MORTALITY VERSUS WATER TGP es CHINOOK SALMON OVER 50 mm. IN LENGTH MORTALITY RANGE - 20 - 70 % TIME TO MORTALITY - Hrs. 400 300 200 100 0 -100 -• • • • • • • • : -• • • -• • • • • • • s • • • • -• • 1 • • • • • • • • "" L • 1 1 • i •I-• • • • • l 1.05 1.1 1.15 1.2 1.26 1.3 WATER TGP - Atms. Note: Negative timet correspond to •xperimentt with no mortality. FIGURE 18: Time to Mortality Versus Water TGP for Chinook Salmon Over 50 mm. in Length. 69 TIME TO MORTALITY VERSUS WATER TGP S O C K E Y E SALMON OVER 50 mm. IN LENGTH MORTALITY RANGE = 20 - 70 % TIME TO MORTALITY - Hrs. 1000 800 600 400 200 0 -200 a s ts ffl (E B S S i ra *!1 anas 1.05 1.1 1.15 1.2 1.25 WATER TGP - Atms. 1.3 1.35 N o t e : N e g a t i v e t i m e s c o r r e s p o n d to e x p e r i m e n t s w i t h no m o r t a l i t y ^ ,. . . . . -rr^ot^ FIGURE 19: Time to Mortality Versus Water TGP for Sockeye Salmon Over 50 mm in Length. 70 indication of a mortality threshold at a TGP of 1.17 Atms. This is particularly evident from the clustering of both positive and negative data near 1.17 Atms. Also apparent at this threshold is a minimum time to mortality of about 100 hours. This is similar to that observed with Chinook salmon as described above. Coho Salmon Greater Than 50 mm.: Shown in Figure 20 is the time to mortality versus water TGP for Coho salmon greater than 50 mm. in length. An aspect of these data is the relative scarcity of information in the vicinity of 1.1 to 1.17 Atms. TGP. It is clear there are mortalities at 1.12 and 1.175 Atms. However, there is nothing to suggest that these are in fact thresholds. Also, as described earlier, the five data sets with water p 0 2 above 271 mmHg. are significantly higher than all other times of mortality shown in the figure. Steelhead Trout Greater Than 50 mm.: For Steelhead trout there are sufficient positive and negative data just at and above a water TGP of 1.15 Atms. to suggest this is a threshold for this species (Figure 21). Again a minimum time to mortality is evident at 1.15 Atms. As water TGP approaches 1.1 Atms., from lower values, the combination of negative times to mortality and the sudden increase to positive times at a TGP of 1.1 Atms. suggest this is the lower mortality threshold. Cutthroat Trout Greater Than 50 mm.: Finally, the data for Cutthroat trout are shown in Figure 22. In this case, there is no information in the database below a water TGP of 1.15 Atms. On the other hand, the data that do exist show a threshold near 1.15 Atms. Once again, there is strong indication of a minimum time to mortality at 1.15 Atms. TIME TO MORTALITY VERSUS WATER TGP COHO SALMON OVER 50 mm. IN LENGTH MORTALITY RANGE • 20 - 70 % 71 1000 800 600 400 200 TIME TO MORTALITY - Hrs. -200 1.05 • • Mi. 1.1 1.15 1.2 1.25 WATER TGP - Atms. 1.3 p02 « 271 mmHg. D p02 > 271 mmHg. Note: Negative times correspond to experiments with no mortality. FIGURE 20: Time to Mortality Versus Water TGP for Coho Salmon Over 50 mm. in Length. TIME TO MORTALITY VERSUS WATER TGP STEELHEAD TROUT OVER 50 mm. IN LENGTH MORTALITY RANGE • 20 - 70 % 72 600 500 400 300 200 100 -TIME TO MORTALITY - Hrs. -100 --• • • • • • • • • • • • • • \ " 1 " • • * - • • • • n -* • - H • • • 8 -• • • • a • i • • • : t j j : : i : i i 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45 WATER TGP - Atms. Note: Negative timet correspond to experimentt with no mortality. FIGURE 21: Time to Mortality Versus Water TGP for Steelhead Trout Over 50 mm. in Length. TIME TO MORTALITY VERSUS WATER TGP 7 CUTTHROAT TROUT OVER 50 mm. IN LENGTH MORTALITY RANGE • 20 - 70 % TIME TO MORTALITY - Hrs. 400 300 200 100 -100 1.1 1.15 1.2 1.25 WATER TGP - Atms. 1.3 1.35 Note: Negative timet correspond to experiments with no mortality FIGURE 22: Time to Mortality Versus Water TGP for Cutthroat Trout Over 50 mm. in Length. 74 4.2.5 EFFECTS OF WATER DISSOLVED OXYGEN TENSION: As described in Section 3, the theoretical thresholds for vascular bubble growth show a dependence on the partial pressure of dissolved oxygen in the water. To search for such a dependency, the database was filtered to restrict the records to those containing information on water TGP, p02, depth and fish length. Fish Less Than 50 mm.: An examination of the records show that, for fish less than 50 mm. in length, the water oxygen partial pressure ranges from 111 to 191 mmHg. These data can be subdivided into dominant groupings based on p 0 2 greater than 170 mmHg. and less than 113 mmHg. The results of this separation are shown in Figure 23. The data records at this level of filtering contain information on Chinook and Coho salmon and Steelhead trout only. In the figure, it is apparent that no distinct relationship exists between time to mortality and water p 0 2 for fish less than 50 mm. in length. There may be a separation of thresholds based on p 0 2 at 1.12 and 1.155 Atms. Unfortunately, there are not enough data in this range of TGP to confirm this separation. Also, it is not clear if the separation is for the same threshold at different levels of p 0 2 or whether there are two separate thresholds. Yet another complexity associated with these data is the effect of depth on mortality thresholds. This is examined further in section 4.2.6, below. Fish Greater Than 50 mm.: The data records show that water p 0 2 ranges from 63 to 458 mmHg. for fish greater than 50 mm. in length. By detailed filtering of these data, the records can be broken into groups based on p 0 2 ranges of, 60 to 90, 111 to 119, 130 to 190, 192 to 221 and 271 to 483 mmHg. These data are shown in Figure 24 where, again time to mortality is plotted as a function of water TGP. Except for the TIME TO MORTALITY VERSUS WATER TGP FOR FISH LESS THAN 50 mm. WATER p02 RANGES AS INDICATED 75 2000 1800 TIME TO MORTALITY - Hrs. 1400 1200 800 600 400 200 0 -200 -• • • f • -+ — • +f--• • • + 4-1 - • • jt • • 1 + • • + i i 1.05 1.1 1.15 WATER TGP " p02w < 113 mmHg. 1.2 Atms. + p02w > 170 1.25 Note: Negative times correspond to experiments with no mortality FIGURE 23: Time to Mortality Versus Water TGP for Fish Less Than 50 mm. TIME TO MORTALITY VERSUS WATER TGP FOR FISH GREATER THAN 50 mm. WATER p02 RANGES AS INDICATED 76 TIME TO MORTALITY - Hrs. 1000 -200 1.1 1.15 1.2 1.25 1.3 1.35 WATER TGP - Atms. 1.4 1.45 • 60-90 0 192-221 • 111-119 + 130-190 x 271-483 mmHfl. Note: Negative time* correspond to experiments with no mortality. FIGURE 24: Time to Mortality Versus Water TGP for Fish Greater Than 50 mm.; Water p 0 2 Ranges as Indicated. 77 five records at water p 0 2 levels greater than 271 mmHg., there is no clear separation of time to mortality or thresholds based on water p 0 2 . However, the difficulty in detecting a relationship has to do with the scale at which the data are being examined. An even finer analysis of the records shows two distinct sets of data that exhibit p 0 2 dependency. The first involves the experiments of Nebeker et al. (1979a). In four experimental series, water TGP was held constant at 1.264, 1.314, 1.364 and 1.414 Atms. while water p 0 2 was varied. The results, this time plotted as time to mortality versus water p 0 2 , are shown in Figure 25. The legend at the bottom of the figure identifies the data associated with each level of TGP. At each TGP it is evident there is an increase in time to 50% mortality with increasing water p 0 2 . The most dramatic increase occurs at the lower TGP of 1.264 Atms. Although the data in this range of TGP show time to mortality is dependent on water p 0 2 , there is nothing to suggest the existence of thresholds. These data and one other set are the only records in the database that show a p 0 2 dependency in time to mortality. Perhaps the most intriguing set of records are those of Rucker et al. (1975). These data are for a water TGP of 1.193 Atms. and water p 0 2 varying between 80 and 483 mmHg. Figure 26 shows the corresponding times to 25% and 50% mortality plotted against water p 0 2 . It is apparent in the figure that time to mortality is less than 220 hours for water p 0 2 levels between 80 and 249 mmHg. As water p 0 2 increases between these two values, the time to 25% and 50% mortality also increases gradually. However, between 249 and 271 mmHg. there is almost a five fold increase in time to 25% mortality. The 50% level of mortality was not reached in an exposure TIME TO 50% MORTALITY VERSUS WATER p02 STEELHEAD TROUT: WATER TGP AS INDICATED DATA OF NEBEKER et al. (1979a) 78 Time to Mortality - hrs. 22 100 200 300 400 500 Water p02 - mmHg. • TGP • 1.264 Atms. + TGP - 1.314 Atms. * TGP • 1.364 Atms. • TGP • 1.414 Atms. FIGURE 25: Time to 50 % Mortality Versus Water p 0 2 for Steelhead Trout: Water TGP as Indicated. TIME TO MORTALITY VERSUS WATER p02 COHO SALMON: WATER TGP • 1.193 Atms. DATA OF RUCKER, 1975 79 1000 900 800 700 600 500 400 300 200 100 TIME TO MORTALITY - Hrs. -100 + + B + _0_ 0 100 200 300 WATER p02 - mmHg. 400 D 50% Mortality + 25% Mortality Note: All fish greater than 60 mm. Negative times correspond to no observed mortality in 1000 hrs. FIGURE 26: Time to Mortality Versus Water p 0 2 for Coho Salmon: Water TGP 1.193 Atms. 80 period of 1000 hours once water p 0 2 rose above 249 mmHg. (Negative times in the figure). Nor was a 25% mortality reached during the experiment at water p 0 2 levels above 300 mmHg. This behavior strongly suggests the existence of a TGP threshold at 1.193 Atms. that is dependent on water p 0 2 . Further, the threshold is located between a water p 0 2 of 249 mmHg. and 271 mmHg. It is this response that made these particular data unique as pointed out early in this analysis. 4.2.6 COMPENSATION DEPTH: As discussed in Section 3, theoretical thresholds for all forms of bubble growth should increase with increasing water depth. If there is sufficient depth, and fish use that depth, the effects of supersaturation can be avoided or reduced. The data of Shrimpton, Randall and Fidler (1988) show that small fish exposed to supersaturated water seek depth to overcome excess buoyancy induced by swimbladder overinflation. Because large fish do not experience the same degree of over buoyancy as small fish (Shrimpton, Randall and Fidler, 1988), they do not seek depth as a means of compensating for supersaturation. In order to examine this effect, the database total gas pressures were corrected for depth. That is, TGP was reduced by the hydrostatic head reported in the data. The correction formula used is: T G P c o r r . = T G P uncorr . - ( 7 3 - 1 • h / pAtms.) where, TGP is in atmospheres, PAtms. i s ' n m m H 9 - and h is in meters. The constant, 73.1, combines the density of water and the gravitational constant to yield units of mmHg./m. The results of this correction are shown in Figure 27a for fish less than 50 mm. The correction produces a horizontal shift of the data to the left on the TGP axis (See 81 Figure 23 for a comparison of the corrected data with uncorrected data). With the correction of TGP for depth, the apparent lower threshold is shifted from 1.12 Atms. to 1.1 Atms. It will be recalled from previous discussions that this corresponds to the lower threshold of mortality for fish greater than 50 mm. The same correction was applied to records for fish greater than 50 mm. An inspection of Figure 27b again shows a leftward shift of data; however, no other changes appear as a result of the depth correction (See Figure 24 for a comparison of the corrected data with uncorrected data). Based on the work of Shrimpton, Randall and Fidler (1988), a correction of TGP for depth may not be appropriate for fish greater than 50 mm. The database was further, analyzed for evidence of depth compensation effects. Although there were clear indications of depth compensation by fish, the results were not conclusive because of species variations and unknown levels of water pG^. Figure 28 shows the best correlation that was obtained and is restricted to fish greater than 50 mm. in length. In the figure, water pO>> is unknown for all data at a depth of 0.61 meters. With this in mind, the data suggest that depth leads to increased survival time at the same level of TGP. In the data records, depths range from essentially zero to about four meters. In most cases there is no restriction on the movement of fish within the depth indicated in each record. The one exception is the data of Knittel et al. (1980). In these experiments Steelhead trout were exposed to supersaturated water in cages held at specific depths. TIME TO MORTALITY VER8U8 WATER TGP FOR FISH LESS THAN 60 mm. WATER TOP CORRECTED FOR DEPTH 82 1600 1600 1400 1200 1000 600 600 400 -200 -0 -200 TIME TO MORTALITY - Hr«. + + 105 11 116 12 126 WATER TOP - Atms. 13 136 14 p02w - 112 170-192 mmHg. wZV&ZiTZZ^F* FIGURE 27: Time to Mortality Versus Water TGP for Fish Less Than 50 mm.: Water TGP Corrected for Depth. TIME TO MORTALITY VERSU8 WATER TGP FOR FISH GREATER THAN 60 mm. WATER TGP CORRECTED FOR DEPTH 1000 TIME TO MORTALITY - Hr«. 106 116 12 126 WATER TGP - AtlTM. 13 136 14 p02w< 80 rn-ti7 0 • 178-183 ° »«70 mmHg. Not* Naeatlw time* eermpond to •xporlatanta with no Mortality. FIGURE 27: Time to Mortality Versus Water TGP for Fish Greater Than 50 mm.: Water TGP Corrected for Depth. TIME TO MORTALITY VERSUS WATER TGP FOR FISH GREATER THAN 50 mm. Chinook • 1, Coho • 2, Steelhead • 4 83 TIME TO MORTALITY - Hrs. 350 300 250 200 150 100 50 -* * X • • 1 1 1.17 1.175 1.18 1.185 1.19 1.195 WATER TGP - Atms. • 1 - (.60M) + 1 - (.26M) * 2 - (.60M) D 2 - (.14M) * 4 - (.60M) 0 4 - (.26M) Depths as Indicated in parentheses. Chinook and 8teelhead at 60 % mortality Coho at 20% mortality FIGURE 28: Time to Mortality Versus Water TGP for Fish Greater Than 50 mm.; Chinook, Coho and Steelhead Trout. TIME TO 50% MORTALITY VERSUS WATER TGP WITH AND WITHOUT TGP DEPTH CORRECTION STEELHEAD DATA OF KNITTEL ET. AL. (1980) 84 TIME TO MORTALITY - Hrs. 50 40 30 20 10 -• • • > • f p JL • * • • B • B 1.1 1.15 1.2 1.25 1.3 1.35 1.4 WATER TGP - Atms. 1.45 • CORRECTED TGP • UNCORRECTED TGP FIGURE 29: Time to 50 % Mortality Versus Water TGP With and Without TGP Depth Correction: Steelhead Trout. 85 TGP ranged from about 1.19 Atms. to 1.41 Atms. Figure 29 shows the results of these experiments in terms of time to 50% mortality versus water TGP. The solid points correspond to data uncorrected for depth while the open squares are for TGP corrected for depth. It is clear that depth correction significantly reduces the scatter in the data and suggests a hyperbolic or asymptotic form of response. Further, the corrected data show a TGP threshold near 1.19 Atms.. This is well above that indicated in the rest of the Steelhead data (Figure 21). However, water p 0 2 is unrecorded in the data of Knittel et al. (1980). 4.3 DISCUSSION: From this analysis, it is clear that the response to supersaturated water by Chinook, Coho and Sockeye salmon along with Steelhead and Cutthroat trout is highly dependent on fish size. The difference in time to mortality between fish greater than 50 mm. and those less than 50 mm. is so strong as to suggest a discontinuous relationship between time to mortality and fish size. This would imply yet another threshold that must be considered when mathematically modeling time to mortality relationships. In an analysis such as that used by Jensen et al. (1985), which depends on continuous functions for regression procedures, ignoring such a threshold can severely distort the formulation of the model. Clearly, the solution to this problem is to separate the data based on size and apply the model to the data sets independently. However, this would not solve all problems associated with multiple thresholds. As pointed out in the introduction to this thesis, if there are multiple thresholds dependent on water TGP and p 0 2 , further separation of the data would be necessary before the models of Jensen et al. (1985) could be used successfully. The preceding database analysis strongly suggests the existence of more than a single threshold for mortality in fish exposed to supersaturated water. 86 It is interesting to compare the predicted response of the models of Jensen et al. (1985) with the data upon which the models are based. Figures 30 and 31 show this comparison for their Models 1 and 15 respectively. In Figure 30, where predicted time to 50% mortality is plotted versus TGP, the model describes the data reasonably well only at TGP levels above 1.22 Atms. Between a TGP of 1.15 and 1.22 Atms., the predicted response diverges from the bulk of data in this region. Below a TGP of 1.15 Atms., there is considerable variation between the model and experimental data. In Figure 31 the model predictions are obtained by using the TGP and oxygen to nitrogen ratio of each experimental datum point to predict time to 50% mortality. Again, above a water TGP of 1.25 Atms., the data and model predictions are relatively similar. However, below this level of TGP there are significant differences between the two. Except for the one point at 1182 hours, the model consistently over predicts time to mortality for TGP levels between 1.1 and 1.17 Atms. Between 1.17 and 1.25 Atms., the model tends to under-predict time to mortality. Although not conclusive for all species examined, the database analysis suggests that two, TGP related, mortality thresholds may be involved in the response of fish to supersaturation. A lower threshold is clearly apparent at a TGP of about 1.1 Atms., while a second threshold may exist in the TGP range of 1.15 to 1.17 Atms. The evidence for the two thresholds is strongest for Chinook salmon greater than 50 mm. in length (Figure 18). The evidence for a higher threshold near a TGP of 1.15 Atms. is particularly clear for Sockeye salmon and Cutthroat trout (Figures 19 and 22 respectively). The data also suggest that the higher threshold varies slightly with fish species. Model 1 of Schnute and Jensen 1986 Compared with Dataset 1 87 Time to 50% Mortality - hrs. 1400 i 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 TGP - Atms. Model + Dataset 1 FIGURE 30: Model 1 of Schnute and Jensen 1986 Model 15 of Schnute and Jensen 1986 Compared with Dataset 2 88 Time to 50% Mortality - hrs. (Thousands) 1.2 i q r 0.2 -1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 TGP - Atms. •=> Model + Data FIGURE 31: Model 15 of Schnute and Jensen 1986 89 For fish less than 50 mm. in length the only mortality threshold that is apparent, is one at a TGP of 1.12 Atms. However, if depth corrections are applied to this threshold (assuming small fish seek compensation depth), the threshold for zero depth is shifted to 1.1 Atms. (Figure 27a). This places the apparent lower threshold at about the same TGP as for fish greater than 50 mm. with TGP uncorrected for depth. Although there is evidence for two TGP thresholds for fish greater than 50 mm. in length, the physiological consequences of these thresholds is not clear. Nor is it clear which lethal symptoms are associated with each threshold. As discussed earlier, the data for Chinook salmon greater than 50 mm. in length suggest two separate mortality mechanisms are operating over two different ranges of TGP. Furthermore, at a TGP of 1.17 Atms., there appears to be a transition from one mechanism of mortality to another. This is accompanied by a dramatic increase in time to mortality. It is not clear what these mechanisms are or the reasons for the apparent transition from one to the other. However, the experimental work of this thesis yields further insight into these mechanisms and the nature of the apparent transition. These results are examined in Section 5 of this thesis. An indication of the cause of mortality at the higher TGP threshold is suggested in the studies of Stroud and Nebeker (1976) on Steelhead trout. In this work, symptoms of GBT were recorded for fish exposed to various levels of water TGP. It was noted that vascular bubbles in the gill arteries did not appear until water TGP rose above 1.15 Atms. Meekin and Turner (1974), made similar observations; however, they reported only dissolved nitrogen concentrations and not TGP. Assuming the apparent threshold at 1.15 Atms. is associated with vascular system bubble growth, the data from Table III, Appendix C, that contain information on water p 0 2 can be plotted in 90 relation to the theoretical bubble growth thresholds (Section 3). This is shown in Figure 32. The data shown as "Other" in the figure correspond to all data in the database between a water TGP of 1.153 and 1.176 Atms. for which water p02 is known. In addition, the thresholds indicated in the data of Rucker (1975) are shown. Interestingly, the critical nucleus radius indicated by this cross plotting is on the order of 12 to 14 fjM. This radius is about twice the size of erythrocytes for these species and also about twice the size of tissue capillaries. This would imply that the formation of bubbles from nuclei in the vascular system occurs in vessels somewhat larger than capillaries. As was shown in Section 3, where the variation in vascular system pressure was examined, this result would be consistent with the hypothesis that the arterioles just upstream of the capillary tissue beds are the most likely location for bubble formation. Of the 528 data records containing information on water p02, only 77 records (those of Rucker, 1975 and Nebeker et al., 1979a), yield relationships between time to mortality and water p 0 2 (Figures 25 and 26). The absence of correlation in the other data is probably due to the wide variation in water TGP in the database combined with a rather sparse range of water p02 values. Another complicating factor may be the depth at which fish position themselves in the water column. For example, Dawley et al. (1976) found that groups of 40 mm. Chinook salmon fry and 180 mm. Steelhead trout responded to increasing levels of supersaturation by increasing their depth in the water column. However, Steelhead trout held lower positions in the water column than did Chinook salmon for the same TGP. This contrasts somewhat with the data of Shrimpton, Randall and Fidler (1988). These data show that the depth single Rainbow trout fry seek in the water column is proportional to water TGP. 91 TGP THRESHOLDS FOR BUBBLE GROWTH IN ARTERIAL BLOOD AS A FUNCTION OF WATER p02 AND NUCLEUS RADIUS TGP THRESHOLD - Atms. 1.29 1.07 50 Nucleus Radius 6 Meters * 10 100 150 200 250 300 350 WATER P02 - mmHg. A Other Data Rucker (1975) Water Depth • 0.0 M. F • 0.79 Water Temp • 5 - 1 5 deg. C. Atmospheric Pressure • 760 mmHg. FIGURE 32: TGP Thresholds for Bubble Growth in Arterial Blood. 92 Thus, there may be behavioral characteristics based on species differences and the number of fish that modifies the depth compensation response. The effect of depth on time to mortality is clearly shown in the data of Knittel et al. (1980). By correcting TGP for depth, a much stronger correlation of time to mortality with water TGP is obtained. The hyperbolic or asymptotic form of response indicated in the corrected data is typical of dose response relationships commonly used in toxicology studies (Sprague, 1969 and Warren and Doudoroff, 1971). The form is also consistent with the models used by Jensen et al. (1985). However, the data of Knittel et al. (1980) correspond to TGP levels at or above the upper TGP threshold indicated in the database. Thus, there data is for a single threshold. Although the correction of TGP for fish restrained to specific depths appears valid, it is seldom that such restrictions can be achieved. 5.0 EXPERIMENTAL STUDIES 93 5.1 INTRODUCTION: A theoretical model for vascular system bubble growth was reviewed and expanded in Section 3. The model predicts that the growth of vascular system bubbles is dependent on TGP thresholds. Furthermore, these thresholds were shown to be a function of water p02, water depth, barometric pressure and the effective size of nucleation sites in the vascular system. The principal unknown in the model is the effective size of the nucleation sites. If this can be established experimentally, Equation 4 will offer a complete description of thresholds for this type of bubble growth. It was hypothesized in Section 3 that the growth of bubbles in the vascular system might lead to alterations in vascular system pressure. Because the cardiovascular system is a relatively closed fluid system, pressure should increase as a result of the gas volume added by the growing bubbles. This type of response was modeled mathematically as presented in Equation 11 and shown in Figures 8 and 9. If such a perturbation can be detected experimentally, it would serve as an indicator of bubble growth thresholds. Once thresholds are defined, the effective radius of cardiovascular nuclei can be back calculated from Equation 4. Also reviewed in Section 3 was the mathematical model for bubble growth in the environmental water or for sub-dermal bubble growth on the external body of fish exposed to supersaturated water. The model predicts that, for nucleation sites comparable in size to those of the vascular system, bubble growth will occur at lower 94 TGP thresholds than those of the vascular system. However, it is not known if this form of bubble growth can produce mortality in fish. In any case, there is an experimental need to verify the threshold equation for these types of bubble growth. In the preceding section an analysis of data from the literature implied there are may be two thresholds for mortality in fish exposed to supersaturated water. The higher threshold, in the vicinity of 1.15 to 1.17 Atms. TGP, appears to correlate with other experimental findings regarding the appearance of intravascular bubbles in gill lamella (Stroud and Nebeker, 1976). This and other threshold correlations, plotted in Figure 32, show that if the higher threshold is associated with intravascular bubble growth, the effective size of the nuclei involved is on the order of 12 to 14 /L/M. in radius. A lower threshold, in the vicinity of 1.1 Atms., also appears in the literature database. If this threshold is associated with bubble growth in the environmental water or sub-dermal bubbles on the skin of the animal, the effective size of nucleation sites is about 12/LiM. Thus, theory predicts there are multiple thresholds for bubble growth in fish exposed to supersaturated water. Data from the literature suggest the existence of two thresholds for mortality in fish exposed to supersaturated water. However, the two are uncorrelated. That is, there is no clear association of the theoretical bubble growth thresholds and the observed thresholds for mortality. Mortality may be caused by bubble growth. However, other physiological insults resulting from supersaturation may also be responsible. Thus, there is a need to experimentally study the relationship between thresholds for bubble growth and physiological parameters that may establish whether or not bubbles are responsible for death in 95 fish. The experimental approach to this problem was accomplished in two distinct experimental phases. 5.1.1 PHASE I EXPERIMENTS: The Phase I experiments examined the response of the vascular system to intravascular bubble growth. The purpose was to determine if detectable increases in blood pressure accompany bubble formation and growth. If an increase in pressure is observed, it can be used as an indicator to experimentally search for bubble growth thresholds. 5.1.2 PHASE II EXPERIMENTS: The Phase II experiments involved examining an array of other physiological parameters as indicators of thresholds for bubble growth. In addition to intravascular bubbles, these experiments focused on thresholds for water bubble and sub-dermal bubble growth. In particular, arterial p02, pH, hematocrit, and catacholamine levels were surveyed. These were correlated with the severity of GBT symptoms and water TGP and pC^. Accompanying these experiments was a study of respiratory performance under conditions of dissolved gas supersaturation. 5.2 EXPERIMENTAL MATERIALS AND METHODS The section that follows contains a description of the materials and methods used in both phases of experimental work. The descriptions are of a general nature and common to many of the experiments. For example, the methods of cannulating fish and the device used to produce supersaturated water are common throughout both phases of experimental work. As each experimental series is examined in detailed, unique procedures associated with that series will be described. 96 5.2.1 EXPERIMENTAL APPARATUS AND WATER PARAMETERS Production of Supersaturaterd Water: A packed column was used to produce supersaturated water for the Phase I and Phase II experiments. This device employs turbulent mixing of gas and water phases under pressure to accomplish the necessary gas exchange (Fidler 1983). The column consisted of a plastic pipe, 2.5 meters long by 200 mm. inside diameter, filled with 1.5 cm. Norton Intalox saddles. The Intalox saddles produce turbulence in water that falls through the column, thereby enhancing the gas exchange process. The column was mounted in a vertical position and sealed at both ends except for water and gas ports. Water supply to the column consisted of two independent flows. The main supply entered at the top of the column and fell over the packing material while a continuous flow of air or pre-selected gas mixtures passed upward through the packing. The second supply, or make-up water, entered at the bottom of the column and mixed with the outflow supply of supersaturated water. A float valve at the foot of the column controlled make-up water flow and assured a uniform discharge from the column. The make-up supply also allowed fine tuning of the system Total Gas Pressure. Pressure regulators upstream in all raw water supply lines insured uniform inlet water flow to the system. The gas supply to the column was either atmospheric air under pressure or a pressurized mixture of oxygen, nitrogen and water vapor. A three way valve in the gas supply system allowed switching between air and the pre-selected gas mixtures. The mixture ratio of oxygen to nitrogen was controlled by a gas flow mixing system with a p 0 2 monitoring control loop. When the gas mixer was used, a Radiometer E5046 thermostated oxygen electrode measured p 0 2 in the outflow water from the 97 column. The monitored electrode signal was used in an electronic feedback system to regulate gas mixture to the column. The setpoint was a pre-selected p 0 2 level fixed by a calibrated potentiometer on the controller. A pressure cooker type relief valve at the top of the column controlled pressure within the column. Column pressure could be maintained at a precise level by adjusting the weight of the relief valve. With this system, it was possible to regulate Total Gas Pressure and dissolved oxygen concentration within narrow tolerances for long periods. Water from the column flowed into a mixing chamber and then into a head tank. The surface of the water in the head tank was covered with a styrofoam float to reduce dissolved gas loss to the atmosphere. Water for the experiments was drawn from the head tank and introduced into live boxes that contained the experimental animals. Water pH, temperature, total gas pressure and p 0 2 were measured in the head tank at a point near the discharge to the live boxes. The general arrangement of the packed column and other experimental apparatus are shown in Figure 33. As shown in the figure, a separate supply of air equilibrated water was available for holding fish in the live boxes before the supersaturation experiments. Figure 34 shows an individual live box with a fish in place. The depth of the box was no more than 10 cm. and the box was open to the atmosphere to prevent hydrostatic head buildup. Water Dissolved Gas Measurements: Throughout the Phase I and Phase II experiments, water dissolved gas tensions were determined by measuring Total Gas Pressure and p 0 2 . TGP was measured continuously with a Model 300 Nova Tech Saturometer. Before each experiment the instrument output was set to zero and barometric pressure recorded. All subsequent measurements of TGP were corrected for changes in barometric pressure occurring during the experiment. 98 PRESSURE REGULATOR AIR SUPPLV SI PACKED COLUMN mm OXVGEN ELECTRODE MIXING CHAMBER GAS MIXTURE CONTROLLER GAS CYLINDERS WATER SUPPLV BAROMETRIC PRESSURE RADIOMETER PHM-71 PRESSURE TRANSDUCER PH ELECTRODE V OXVGEN ELECTRODE \ A A A A A . j SATUROMETER I THERMOMETER OLIVETTI M24 COMPUTER / DATA TRANSLATION DATA ACQUISITION CARD SVRINGE — a CANNULA IN DORSAL AORTA HEADER TANK LIVE BOX EXPERIMENTAL APPARATUS FIGURE 33: Experimental Apparatus 99 FIGURE 34: Live Box 100 The sensor portion of the instrument was placed in the head tank supplying water to the live boxes (Figure 33). The sensor was shaken continuously during experiments by a mechanical shaker. This prevented bubble formation on the silastic tubing membrane of the instrument which has been shown to reduce instrument sensitivity (Bouck, 1982). Dissolved oxygen was monitored continuously by passing a stream of water from the head tank through a Radiometer Model E5046 oxygen electrode. The electrode was housed in a water jacket which maintained the electrode at the experimental water temperature. The electrode electrical output was connected to a Radiometer PHM 71 signal conditioner and meter. The system was calibrated before and at times during each experiment with two standards. The low standard consisted of water that had been completely degassed of oxygen. The high standard was water that had been equilibrated with atmospheric air. The high standard calibration was corrected for the partial pressure of water vapor at the experimental temperature and barometric pressure at the time of calibration. Water pH and Temperature: Water pH was measured with a Radiometer G279/G2 glass capillary electrode and F497 calomel electrode connected to a Radiometer PHM 71 signal conditioner and meter. Before and following each experimental series, the pH electrode was calibrated using Radiometer precision phosphate buffers S1500 and S1510. Water pH was measured at prescribed intervals during each experimental series. Water temperature was measured with a mercury thermometer. 5.2.2 COMPUTERIZED DATA ACQUISITION SYSTEM: As shown in Figure 33, most experimental measurements were recorded digitally on a personal computer. A Data 101 Translation DT-2801 analog to digital card, installed internally in the computer, interfaced with the computer main bus. The DT-2801 card converts voltage signals from the various instruments into digital data which were recorded on magnetic media by the Olivetti M24 personal computer. The data collection process and operation of the system was managed by the Laboratory Technologies LabTech Notebook computer program. This software allows complete control of data sampling rates and experimental protocol. Data were stored on floppy diskettes for later analysis with the Lotus 123 spreadsheet computer program. 5.2.3 EXPERIMENTAL ANIMALS AND SURGERY PROCEDURES Experimental Animals: Rainbow trout (Salmo gairdneri), weighing between 220 and 850 grams were obtained from West Creek Trout Farms of Aldergrove British Columbia. The fish were maintained in outdoor tanks at water temperatures ranging from 5 to 15° C , depending on the time of year. The animals were fed once weekly, ad libitum, a diet of commercial trout food. When experimental temperatures differed from holding temperatures, fish were acclimated to the experimental temperature by temperature changes of no more than 0.5 °C. per day. The maximum temperature re-acclimation required during any experimental series was 4° C , (i.e. from 5 °C. to 9° C) . Surgery Procedures: For experiments requiring the measurement of blood parameters, fish were fitted with dorsal aorta cannula. Surgical implantation of the cannula was accomplished with animals anesthetized in a pH buffered (pH = 7.5) solution of tricaine methane sulphonate, MS-222, at a concentration of 1:10,000. Cannulation was performed on a surgery table where the gills of the fish were 102 continuously irrigated with an oxygenated, half concentration dose (1:20,000) of the anesthetic solution. The temperature of the irrigation solution was the same as the holding temperature for the fish. Cannulation was performed using the technique of Sovio et. al. (1972), in which a polyethylene tube (PE 50) is guided into the dorsal aorta by way of a sharpened steel wire within the cannula. Using a surgical 20 gage needle, a blind puncture is made centrally in the roof of the mouth between the first and second gill arches. The cannula, with the steel wire protruding 3 mm. from the tip, is guided through the puncture and forced through the wall of the aorta. Once in the aorta the wire is slowly withdrawn from the PE tubing while the tubing is fed into the aorta for a distance of 30 to 70 mm depending on the size of the fish. When in place, the cannula is anchored with sutures to the roof of the mouth at two locations. The cannula is then led through a flanged PE 200 sleeve which had been previously forced through the roof of the mouth just ahead of the nares. The PE 200 sleeve is anchored with cotton thread looped around the sleeve and tightly knotted next to the skin where the sleeve exits from the fish. When not in use for blood sampling or pressure measurements, the end of the cannula was plugged with a straight pin to prevent blood loss. During recovery from surgery, cannula were flushed daily with Cortland saline (Wolf, 1963) containing 10,000 USP units/L of sodium heparine. Following surgery, animals were allowed a recovery period of at least 24 hours before being subjected to an experimental procedure. Before committing an animal to an experimental series, blood p 0 2 was measured. If the arterial p 0 2 was not above 90 mmHg., the animal was not used. This was a precaution against failure of the animal to fully recover from surgery and as a screen against animals of poor health. 103 5.2.4 PHYSIOLOGICAL PARAMETERS: Every effort was made to minimize net blood loss by the animals as a result of sampling procedures. For example, when blood was remove for p 0 2 analysis it was returned to the animal following the measurement. Except for hematocrit and pH samples, any blood that was permanently removed from the animal was replaced with an equal volume of Cortland saline. The need to minimize blood removal was considered important in order to obtain accurate hematocrit assays and to minimize any effect of sampling on the intravascular bubble growth process. Blood Pressure: Blood pressure was monitored by connecting the dorsal aorta cannula to a Statham Model 50-B pressure transducer. The transducer was calibrated with a mercury manometer before and following each experiment. The range of calibration was 0 to 100 mmHg. Before connection to the cannula, the transducer pressure chamber and all connecting tubing were filled with Cortland saline containing 10,000 USP units/L of sodium heparine. Three way valves, located on the pressure and vent ports of the transducer, allowed flushing of the system with heparinized saline to remove any bubbles that formed in the tubing or transducer. Care was taken to locate the transducer strain gage diaphragm at about the same level as the lateral line of the fish. This insured that the recorded pressure was the dorsal aorta pressure without hydrostatic components related to elevation differences. The electrical output of the transducer was connected to the computerized data acquisition system. The digitized pressure data were recorded on floppy diskettes for subsequent analysis with the Lotus 123 spreadsheet program. 104 Partial Pressure of Oxygen in Arterial Blood: Arterial blood p 0 2 was measured with a Radiometer E5046 oxygen electrode connected to a Radiometer PHM 71 signal conditioner. The electrode was contained in a housing thermostated to the experimental water temperature. Two techniques were used to draw blood through the electrode. During experiments involving blood pressure measurements, a three way valve in the dorsal aorta cannula allowed blood to bypass the pressure transducer. This blood was drawn through the p 0 2 electrode by way of a peristaltic roller pump. Once the p 0 2 measurement was complete, the pump was reversed and blood returned to the animal. For all other experiments, blood was removed from the animal through the cannula using a 1 ml. syringe. The syringe was then connected to the inlet port of the oxygen electrode system and blood forced through the electrode. The outlet port of the electrode was connected to a long loop of PE 90 tubing that had been previously filled with Cortland saline. Once the measurement was complete, the blood was withdrawn from the electrode and PE 90 tubing loop back into the sampling syringe. The blood was then returned to the animal via the cannula. Arterial Blood pH Measurements: Blood pH was measured with a Radiometer G279/G2 glass capillary pH electrode and K497 calomel electrode thermostated to the experimental water temperature. The electrical output of the electrode was connecter to a Radiometer PHM 71 signal conditioner and meter. Blood from the cannula was drawn into a 1 ml. syringe as described above for measurement of p 0 2 . Before blood in the PE 90 loop was returned to the sampling syringe, the syringe was disconnected from the p 0 2 electrode momentarily and blood drawn directly from the syringe into the pH electrode sampling loop. The syringe was re-connected to the 105 pC"2 electrode port while the pH was being measured. Blood in the pH loop, about 0.05 ml., was not returned to the animal. Arterial Blood Hematocrit: Hematocrit was determined by drawing blood samples into microhematocrit tubes. The samples were taken by sampling directly from the dorsal aorta cannula immediately after blood had been drawn into the pH/p02 sampling syringe. The net blood loss associated with the hematocrit measurements is estimated to be 0.05 ml. per measurement. 5.2.5 PHASE I EXPERIMENTS: The first experimental series involved a survey of the dorsal aorta pressure during exposure to supersaturation. The acquisition of data involved sampling data at various rates depending on the response of the animal. Two sampling sequences were employed. A fast rate, 30 samples per second, gave clear definition of the system pressure pulses. From this, a mean blood pressure and pulse pressure amplitude could be determined. This sampling rate also allowed precise definition of heart rate. Due to computer storage and memory limitations during analysis, this level of monitoring could be maintained for no more than 3 minutes. For longer periods of monitoring, a sampling rate of one sample every 10 seconds was used. The lower sampling rate allowed data recording for up to 15 hours. During these experiments, total gas pressure was varied between 1.1 and 1.3 Atms. while oxygen partial pressure ranged from 100 to 225 mmHg. In addition to measuring blood pressure, blood samples were drawn periodically for dissolved oxygen and hematocrit measurements. For this series of experiments, fish were exposed to supersaturation and monitored individually or in pairs. 1 0 6 In most cases heart rate could be calculated directly from the blood pressure recordings taken at 30 samples per second. In other cases, presumably as a result of bubble formation in the vascular system, pulse pressures were erratic and distorted; thereby making heart rate difficult to determine. In these cases, the Fourier transform capability of the Labtech Notebook program was used to transform the time domain pressure data to frequency domain data (See Champenny, 1971 and Rabiner and Gold, 1975 for a discussion of Fourier transforms and power spectral density analysis). This procedure allowed examination of the pressure traces for the frequency content and principal modes. 5.2.6 PHASE II EXPERIMENTS: The Phase II studies involved several experimental series. Each series had individual objectives and correspondingly different experimental techniques for accomplishing these objectives. Series 1 through 5. Correlation of Physiological Measurements with GBT Symptoms: The objective of experimental series 1 through 5 was to correlate physiological data with bubble growth thresholds and mortality. During exposure to supersaturated water, blood p02, hematocrit and pH were monitored in groups of 12 fish (six cannulated and six un-cannulated). The five series consisted of exposure to water TGP levels of 1.10, 1.12, 1.15, 1.17 and 1.19 Atms., and corresponding p 0 2 levels of 170, 175, 183, 195 and 201 mmHg. An additional component of these studies involved evaluation of the severity of GBT symptoms at death in each animal. This evaluation included an assessment of severity for: 1. ) Extra corporeal bubble formation in the gill lamella. These were bubbles observed in the water between gill lamella and were clearly not internal to the animal. 2. ) Intravascular bubble formation in gill lamella. These bubbles were located in the filamental arteries of the gill lamella and formed within the blood medium. 3. ) Sub-dermal bubble formation in the buccal cavity. These bubbles, as well as the two forms listed below, were obvious blisters that forced separation of the epithelium tissue layer from the underlying tissue. 4. ) Sub-dermal bubble formation on the opercula. 5. ) Sub-dermal bubble formation on the fins. In order to obtain a relative evaluation of symptom severity, an arbitrary scale ranging from 0 to 3 was used. A value of 0 indicates that the particular symptom was absent. A value of 3 indicates the symptom was of the maximum severity observed. As the results of the experiments are examined, photographic examples will be used to illustrate the severity of various symptoms. Visual examinations were made for external symptoms such as sub-dermal blisters on the body, fins and in the mouth. 1 0 8 The examination of extracorporeal gill bubbles and intravascular gill bubbles was done microscopically. The microscopic studies involved excising 4 to 6 samples of gill tissue from each side of the animal, (8 to 10 samples total per animal). The samples were placed on glass slips that had been cooled to the experimental water temperature. The samples were covered with a cooled glass slip and quickly placed under the microscope for examination. The purpose of cooling the glass slips and covers was to prevent bubble formation due to temperature changes. A Wild dissecting microscope equipped with a Leitz photographic system was used to examine and photograph gill and other tissue samples. Series 6. Catacholamine Assays: The objective of this experimental series was to determine if fish exposed to supersaturated water exhibited symptoms of stress as indicated by blood catacholamine levels. Catacholamine assays were conducted on cannulated fish in which no other blood measurements were made. Only fish that were 500 grams or more were used for these assays. Since repeated samples were needed and each sample was at least 700 jul., it was anticipated that the use of large fish would minimize the effects of blood sampling on bubble formation. For each blood sample taken, an equivalent volume of Cortland saline was returned to the animal to minimize total blood loss. Blood samples were placed in 1.5 ml. vials and cooled in an ice bath. Within five minutes the samples were centrifuged to separate plasma from red blood cells. A minimum of 200 /il. plasma was siphoned into 1 ml. vials. These samples were immediately frozen with liquid nitrogen and stored at a temperature of -80° C. until analysis. Analysis for adrenaline and nor-adrenaline was performed with a High Precision Liquid Chromatograph (Spectra Physics, Model Sp8700) using techniques described by Woodward (1982) and Primmett et al. (1986). 109 Series 7. Respiration Frequency and Ventilation Volume: Various facets of the Phase I and II experiments indicated that acute hypoxia is a factor in the death of fish exposed to supersaturated water. In this situation, other physiological responses such as respiratory performance would serve as confirming indicators of this conclusion. In order to monitor respiration, three un-cannulated fish were fitted with surgical rubber masks sutured around the mouth and snout as described by Cameron and Davis (1970). Masks were installed under surgery using the anesthetic procedures described earlier. The fish were then installed in van Dam respiration monitoring boxes. The boxes, constructed of clear plastic, are divided into two compartments separated by a plastic partition containing a circular hole and flange system. When a fish was placed in the aft section of the box, the flange system allowed the rubber mask to be clamped such that water could flow from the forward compartment to the aft compartment only by way of the gill ventilation system. The arrangement is shown schematically in Figure 35. With the outflow ports in the two sections of the box at the same level, no differential hydrostatic head exists between the sections. By measuring the rate of outflow from the aft section of the box, the rate of respiratory water flow could be determined. A visual measurement of respiratory frequency permitted calculation of the respiratory ventilation volume. Series A. B. C and 4: Vascular Bubble Growth Threshold Dependency on Water pOo: The objective of this series was to confirm that vascular system bubble growth thresholds are dependent on water p 0 2 . In these experiments, water TGP was held constant at 1.15 Atms., while p 0 2 was varied. Series A and B corresponded to a water p 0 2 of 100 mmHg. Series C was for a water p 0 2 of 125 and Series 4 (the same as Series 4 described above) a water p 0 2 of 183. Groups of 6 un-cannulated fish were used at each series and the time to mortality for each group was monitored. 110 WATER INLET FLANGE PLATES OVER- • FLOW PIPE SURGICAL RUBBER MASK SUTURED AROUND MOUTH AND CLAMPED BETWEEN FLANGE PLATES. OVERFLOW PIPE U f i N D A M B O X FIGURE 35: Fish in van Dam Respiration Chamber I l l The intent was to determine if a bubble growth threshold could be detected similar to that shown in the data of Rucker (1975) but at a different water TGP and levels of p 0 2 . In addition, an examination of gill lamella at death was made to establish if intravascular bubbles were present. 5.3. PHASE I EXPERIMENTAL RESULTS 5.3.1 GENERAL OBSERVATIONS: A total of 27 fish were examined during this series of experiments. Depending on water TGP and the response of the fish, individual experiments lasted as long as 300 hours and as short as 3.5 hours. Although the cannulation and pressure monitoring procedures were easily implemented, obtaining pressure data throughout a complete experiment was often difficult. In general, data could be obtained through the early periods of all experiments. In fact, at water TGP levels below 1.15 Atms., it was frequently possible to obtain pressure recordings for the entire experimental period. However, at TGP levels above 1.20 Atms., cannula become blocked regularly. In many cases it was clear that blood clotting had occurred and was the probable cause of cannula blockage. In most cases, the cannula could not be cleared and pressure measurements were lost. Hence, it was impossible to determine if intravascular bubble growth was modifying blood pressure. The successful recordings, obtained at higher levels of TGP, did show a response indicative of an interaction between bubble growth and system pressure. Of the successful attempts at monitoring blood pressure, the response to bubble growth was quite variable. However, certain characteristics were often repeated within specific ranges of TGP. For example, at levels of TGP above 1.18 Atms., there was usually an increase in blood pressure, followed by death of the animal. Due to 112 the unpredictable occurrence and often short duration of this response, it was difficult to get clear definition of blood pressure by way of detailed recordings (i.e. the 30 samples per second sampling rate). Most records of blood pressure were obtained at the lower sampling rate of one sample per 10 seconds and gave only mean pressure levels. Nevertheless, the lower sampling rate provided clear indication of increases in blood pressure, presumably due to bubble growth. This result is in agreement with the predicted response based on the theoretical model for bubble growth in a closed system, Section 3. Also, following death, a residual blood pressure was frequently observed for TGP levels above 1.17 Atms. That is, blood pressure did not drop to zero following death as was the case for TGP levels below 1.17 Atms. The residual blood pressures were low (generally less than 10 mmHg.); however, they were characteristic of the higher levels of TGP. At TGP levels below 1.15 Atms. only modest increases in blood pressure were observed during the experiments. At the same time, heart rate would usually decrease and only slight increases in hematocrit could be detected. At all levels of TGP above 1.10 Atms., arterial p 0 2 declined during each experiment. In addition, microscopic examination of the gills showed considerable numbers of extracorporeal bubbles growing between gill lamella. These bubbles were clearly in the water phase, external to the gill lamella, and of a size that could conceivably block respiratory water flow through the lamella. At water TGP levels above 1.17 Atms. blood hematocrit rose dramatically during the experiment and reached maximums just before death of the animal. A nearly 85% increase was observed in one animal. As at lower levels of TGP, arterial p 0 2 fell during the experiments and reached a minimum just before death of the animal. Also, as at lower levels of TGP, many extracorporeal bubbles were found between gill lamella. In addition to these bubbles, there was clear evidence of intravascular bubbles in the filamental arteries of the primary lamella. Often, entire primary lamella were blocked with little or no evidence of red blood cells in the arteries of either primary or secondary lamella. Three fish were tested at a water TGP of 1.1 Atms. and p 0 2 levels of 100, 177 and 225 mmHg., for periods ranging from 198 to 250 hours. Although two fish died before the experiment had reached 250 hours of exposure, none of the animals showed any symptoms of GBT. That is, there were no major alterations in blood pressure, arterial p 0 2 or hematocrit that could be related to GBT. For this series of experiments, it was concluded that thresholds did not exist for any form of bubble growth below a water TGP of 1.1 Atms. 5.3.2 RESPONSE OF INDIVIDUAL FISH: Much of the Phase I experimental effort was exploratory in nature. Also, as mentioned earlier, it was often difficult to get continuous recordings of all measured parameters over the entire duration of an experiment. As a result, many of the data for individual fish were incomplete and cannot be compared directly with data from other fish in the experiments. Thus, tabulated data are not presented. However, to illustrate the variability of the results and at the same time point out important features in the overall response, blood pressure traces of six selected fish will be examined. Fish No. 9 (TGP =1.13 Atms.. pOow = 180 mmHg.): The upper plot of Figure 36 shows the blood pressure recorded before and at 50 hours of exposure for Fish No. 114 9. Pre-exposure blood pressure was about 23 mmHg. and heart rate is 48 beats per minute, (BPM). After 50 hours of exposure blood pressure dropped to 20 mmHg. and heart rate has decreased slightly to 42 BPM. Later, as shown in the bottom plot, mean blood pressure rose to about 30 mmHg. at 75 hours; however, heart rate had fallen to 36 BPM. The rise in mean blood pressure began just after 50 hours of exposure. Finally, at 125 hours mean blood pressure is 19 mmHg. and heart rate has dropped to 27 BPM. The animal persisted at this level for another 52 hours and then died. During the experiment, blood p 0 2 remained near the pre-exposure level of 101 mmHg. until the 125 hour measurement. At that time p 0 2 was 75 mmHg. A sample taken a few hours before death showed a p 0 2 of 40 mmHg. Hematocrit fraction changed very little during the experiment. An increase was observed from a pre-exposure level of 0.31 to 0.33 near the end of the experiment. In addition to the vascular system response just described, various external symptoms of GBT were present. At about 125 hours, blisters were well formed on the surface of the opercula and within fin rays. Also, blisters had formed in the lining of the buccal cavity. These blisters became progressively larger with time. Some minor hemorrhaging from external skin lesions was also present. This was observed in all fish that exhibited severe sub-dermal bubble growth, regardless of water TGP. At death there was evidence of extracorporeal bubbles between gill primary and secondary lamella. However, there was no indication of intravascular bubbles or gill damage and blood pressure was zero at death. This general behavior of blood pressure and other symptoms was observed in four fish; two fish exposed to a TGP level of 1.12 Atms. with p 0 2 levels of 178 and 220 mmHg., and two fish at a TGP of 1.14 Atms with p 0 2 levels of 150 and 250 mmHg. FISH 9 DORSAL AORTA BLOOD P R E S S U R E TGP =1.13 Atms.. p02w = 160 mmHg. PRE EXPOSURE 30 HOURS EXPOSURE H 1 1 1 1 1 r-0 10 20 » TIME -FISH 9 DORSAL AORTA BLOOD P R E S S U R E TCP =1.13 Atme., p02w = 180 mmHg. TIME - S^:. FIGURE 36: Fish 9 Dorsal Aorta Blood Pressure. Heart rates, hematocrits and arterial p 0 2 levels varied between the fish both before and during the experiments. The most noticeable early variation in arterial p 0 2 was during first exposure where the p 0 2 reflected the variations in water p 0 2 . In these fish, reductions in heart rate varied both in frequency and the time over which the reduction took place. At the higher levels of p 0 2 , the fall in heart rate did not take place as early as at the lower levels of p0 2 - The other symptoms including extracorporeal bubble growth in the gills and sub-dermal bubble growth on the external surfaces of the fish did not appear to vary significantly with water p 0 2 . FISH NO. 22 (TGP = 1.15 Atms.. pOow = 180 mmHg.): The upper plot of Figure 37 shows blood pressure just before exposure to supersaturated water. Heart rate is uniform at 61 BPM and blood pressure is 28 mmHg. The bottom plot of Figure 37 shows a 2.3 hour record of blood pressure taken after 86 hours of exposure. The monitoring rate is one sample per 10 seconds. It is clear there is little change in mean blood pressure or pulse pressure (the difference between the maximum and minimum pressure). The pulse pressure is about 5 mmHg. Over the next 50 hours mean blood pressure rose to approximately 40 mmHg. and remained there through 200 hours of exposure (upper plot, Figure 38). Beyond 200 hours, blood pressure began to drop accompanied by periodic decreases in heart rate. The lower plot of Figure 38 shows the response at 256 hours, a few hours before death of the fish. Blood pressure was quite variable but on the order of 10 mmHg. Because of the erratic pressure pulse, heart rate could not be determined precisely. A Fourier analysis of this pulse yielded an array of frequencies with 22 and roughly 48 BPM being dominant. Blood p 0 2 again declined during the experiment with a drop from 115 mmHg., pre-exposure, to 45 mmHg. at 256 hours. Hematocrit fraction showed no major variation. FISH NO. 22 P R E — E X P O S U R E BLOOD P R E S S U R E TGP = 1.00 Atms.. p02w = 187 mmHg. 40 -| 1*. 35 -29 -20 -13 -10 -9 -0 H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r-0 2 4 « 8 10 12 14 1« 18 20 TIME - $•<:. FISH 22 BLOOD P R E S S U R E AFTER 86 HOURS TCP = 1.19 Atpii.. p02w s 180 mmHg. 40 - i 39 -20 -19 -10 -9 -0 H 1 1 1 1 1 1 1 r 0 2 4 6 8 (ThouMnch) TIME - $•<:. FIGURE 37: Fish 22; Pre-exposure Blood Pressure Blood Pressure after 86 Hours. FISH 22 BLOOD P R E S S U R E AFTER 200 HOURS l l g TGP = 1.19 Atms.. p02w = 180 mmHg. 4 « (Thousands) TIME -FISH 22 BLOOD P R E S S U R E AFTER 256 HOURS TGP = 1.19 Atms., p02w = 180 mmHg. 20 -j 19 -18 -1 -o H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 -0 4 8 12 1« 20 24 28 TIME -FIGURE 38: Fish 22; Blood Pressure after 200 Hours. Blood Pressure after 256 Hours. External symptoms of GBT included blistering of fin rays, opercula flaps and the lining of the mouth. At death, gills again showed evidence of extracorporeal bubbles but no major gill damage. At death, blood pressure returned to zero. FISH NO. 11 (TGP = 1.18 Atms.. pQo = 195 mmHg.): Fish 11 showed a blood pressure response that was unique to all the fish tested. Figure 39 shows an array of blood pressure traces taken at periodic intervals. The bottom trace is the pre-exposure blood pressure which has a mean value of approximately 33 mmHg., a pulse pressure of 5 mmHg, and a heart rate of 45 BPM. This response changed very little for 73 hours. At 73 hours of exposure, mean blood pressure rose while pulse pressure declined as shown in the 75 hour trace of Figure 39. Heart rate had decreased to 39 BPM at 75 hours. The rise in mean pressure with decreasing pulse pressure continued through 105 hours. At 105 hours the apparent heart rate was considerably elevated from the pre-exposure heart rate. Fourier analysis of the pressure pulse indicated a dominant frequency in the range of 60 BPM, with higher frequency, lower energy modes also present. The trend in increasing mean blood pressure and declining pulse pressure continued through 115 hours to 117 hours (Figure 40). At 117 hours the mean blood pressure is about 57 mmHg. and pulse pressure is on the order of 2.5 mmHg. Heart rate is extremely difficult to assess from the trace. Fourier analysis of the 115 hour record showed quite variable results depending on the period over which the data were analyzed. Frequencies ranging from 45 BPM to over 90 BPM were present. The unusual signature of the pressure pulse beyond 75 hours appears to be the result of an interaction between the compliance of growing bubbles and the fluid dynamics of the vascular system. 50 FISH 11 ARTERIAL BLOOD PRESSURE TGP =1.18 Atms., p02w = 195 mmHg. 4 5 -40 35 J 30 i 25 105 HRS. PRE-EXPOSURE • i — ' i — | — i — i i — i | i i i — i | — i i i — i — | — i 0 5 10 15 20 25 30 35 TIME - Sec. FIGURE 39: Fish 11; Arterial Blood pressure. Pre-exposure, 75 Hours and 105 Hours. FISH 1 1 ARTERIAL BLOOD P R E S S U R E TGP =1.18 A t m s . , p02w = 195 m m H g . TIME - Sec. FIGURE 40: Fish 11; Arterial Blood pressure. Pre-exposure, 115 Hours, 117 Hours and 118 Hours. 122 The general behavior is one of attenuation of lower frequency pressure pulses combined with the development of higher frequency resonant modes. During this period the fish exhibited periods of violent swimming and thrashing as is evident at the beginning of the 115 hour trace. Beyond 117 hours mean blood pressure dropped rapidly until death just short of 119 hours. In Figure 40 the pre-exposure blood pressure trace is included for comparison. Interestingly, the fish showed a residual blood pressure of 7 mmHg. following death. This residual blood pressure was also observed in other fish at higher levels of TGP. At death, there was evidence of blisters on the opercula and in fin rays as well as some blisters in the mouth of the animal. The extent of blistering at death was not as severe as that seen in fish exposed to lower levels of TGP. Areas of the gill showed evidence of intravascular bubbles and extracorporeal bubbles were present between gill lamella. Arterial p 0 2 declined during the experiment from a pre-exposure level of 105 mmHg. to 32 mmHg. at 118 hours. Hematocrit rose from a pre-exposure level of 0.32 to 0.44 at 118 hours. FISH NO. 17 (TGP = 1.20 Atms.. pOo = 208 mmHg.): Fish 17 exhibited a pre-exposure mean blood pressure of about 20 mmHg. with a pulse pressure of about 4 mmHg. Heart rate was 60 BPM and fairly uniform (upper trace, Figure 41). Except for a rise in mean pressure to 24 mmHg., there was little change in other characteristics of the pressure trace through 10 hours (lower trace, Figure 41). Some temporary excursions to lower mean blood pressures were seen after 49 hours (upper trace, Figure 42). At 56 hours, mean blood pressure began to rise and over a period of about 58 hours reached a maximum of 55 mmHg. (lower trace, Figure 42). FISH 17 BLOOD PRESSURE AFTER 10 HOURS TCP = 1.2 Atms.. p02w = 206 mmHg. FIGURE 41: Fish 17; Pre-exposure Blood Pressure and at 10 Hours. FISH 17 BLOOD PRESSURE AFTER 56 HOURS TCP = 1.2 Atms.. p02w - 208 mmHg. 70 - i o H 1 1 1 1 i 1 1 1 1 1 1 • 0 4 8 12 1« 20 24 (Thouvandv) TIME - $•<•. FIGURE 42: Fish 17; Blood Pressure After 49 Hours and 56 Hours. 1 2 5 This was immediately followed by a rapid plunge in blood pressure and death of the fish. A residual blood pressure of about 9 mmHg. existed following death. This fish, like Fish 11, exhibited periods of violent swimming during the rise in blood pressure from 56 hours to death. No external signs of skin blistering were evident in Fish 17. However, there was evidence of intravascular bubbles in the gills and bubbles between gill lamella. Arterial p 0 2 dropped from a pre-exposure level of 125 mmHg. to about 60 mmHg. at 56 hours. Hematocrit rose from a pre-exposure level of .22 to .37 at 56 hours. FISH NO. 6 (TGP = 1.23 Atms.. pOo = 225 mmHg.): Fish No. 6 exhibited the most violent reaction of all fish tested and experienced death in the shortest time. Shown in the lower left side of Figure 43 is the pre-exposure blood pressure trace. The mean pressure is about 27 mmHg., the pulse pressure around 5 mmHg. and heart rate is uniform at about 60 BPM. Mean blood pressure and heart rate changed little in three hours of exposure. However, at three hours, blood pressure began to rise and, in a fifteen minute period, reached the levels shown in the upper trace of Figure 43. At this time, heart rate was on the order of 40 BPM. The fish became extremely violent as evident in the larger blood pressure fluctuations shown in the figure. Although TGP was immediately reduced to 1.0 Atms., the fish did not recover. Before the animal died three hours later, mean blood pressure declined to about 40 mmHg. The experiment was ended before death of the animal, and it is not known if a residual pressure existed after death. Because of the rapid onset of this reaction, blood p 0 2 and hematocrit were not obtained after the pre-exposure levels were measured. There were no signs of external blistering and no effort was made to examine gills for damage or bubble formation. FISH 6 ARTERIAL BLOOD P R E S S U R E TGP = 1.23 Atms., p02w = 225 mmHg. £ l 0 0 q  E E 90; 0 10 20 30 40 50 60 70 80 90 100 TIME - Sec. FIGURE 43: Fish 6; Arterial Blood Pressure. 127 This was the first fish that showed the hoped for response and it was not clear at the time bubbles might be found in gill lamella. FISH 27 (TGP = 1.25 Atms.. pOow = 215 mmHg.): The pre-exposure mean blood pressure of Fish No. 27 was about 25 mmHg., pulse pressure was 5 mmHg., and heart rate was 63 BPM (upper plot, Figure 44). Following 7 hours of exposure, mean blood pressure, pulse pressure and heart rate were unchanged. These parameters continued to remain unchanged for another four hours. At that time, mean blood pressure began to rise. Pulse pressure became erratic with large changes in amplitude and heart rate varied considerably (Figure 45). The overall response was similar to Fish No. 6; however, the onset of this response was considerably slower than that of Fish No. 6. Mean blood pressure reached a maximum after about 12.5 hours and then began a slow decline until death of the animal. Following death there was a residual blood pressure of about 3 mmHg. During this experiment, arterial p 0 2 declined from a pre-exposure level of 126 mmHg. to about 50 mmHg. at 12 hours. However, the major portion of the decline did not occur until after 7 hours. Hematocrit rose from a pre-exposure level of .21 to a maximum of .39 at 12 hours. Other fish tested in the TGP range from 1.2 to 1.3 Atms. showed blood pressure behavior similar to those of Fish No's. 6 and 27. However, some fish showed a response similar to Fish No. 17 where there was little elevation of blood pressure during exposure to supersaturation. A repeatable rise in blood pressure could be obtained only at TGP levels above 1.25 Atms. In all cases, there was a decline in arterial p 0 2 while hematocrit rose. Gill damage and intravascular bubbles were clearly present; however, there was little evidence of skin blistering. That which did appear was minor and occurred on fish which took the longest time to die. FISH 27 BLOOD PRESSURE AFTER 7 HOURS TCP = 1.29 Atms., p02w = 219 mmHg. (Thousand*) TIME - SEC. FIGURE 44: Fish 27; Pre-exposure Blood Pressure and After 7 Hours. FISH 27 BLOOD PRESSURE AFTER 12 HOURS TGP = 1.29 Atms., p02w = 21S mmHg. 0 0.2 0.4 o.e 0.8 1 (Thousands) TIME - $•«. FIGURE 45: Fish 27; Blood Pressure After 12 Hours. 5.4 DISCUSSION OF PHASE I EXPERIMENTS 130 5.4.1 GENERAL RESPONSE TO SUPERSATURATION: These experiments demonstrate that, above certain levels of TGP, major perturbations in vascular system pressure do occur in fish exposed to supersaturated water. It is presumed that these alterations are the result of bubble growth in the vascular system. Other symptoms such as increased hematocrit, intravascular bubble growth in the gills and residual blood pressure following death support this conclusion. In general, the cardiovascular response to supersaturation is highly dependent on water TGP. Below a TGP of 1.15 Atms., there is limited response of the vascular system in terms of elevated blood pressure, increased hematocrit, intravascular gill bubbles or residual blood pressure following death. These observations imply that vascular system bubbles are not present below a TGP of 1.15 Atms. Above a TGP of 1.25 Atms., increases in blood pressure and residual blood pressures following death are clearly present. These symptoms, visible evidence of intravascular gill bubbles and elevated hematocrit support the conclusion that bubbles are present in the vascular system. The large increases in hematocrit are interpreted as being the result of bubbles displacing intravascular water. This phenomena will be examined further in the Phase II experimental results. Between 1.15 and 1.25 Atms. of TGP, response is highly variable; sometimes giving results similar to those at 1.15 Atms. and sometimes similar to those above 1.25 Atms. Because of the large variability in the blood pressure response, it is not a precise indicator of bubble growth thresholds. The one clear result is that, for adult fish, no threshold for bubble growth exists below a TGP of 1.1 Atms. 131 Other parameters measured during the experiments yield additional information regarding the overall physiological response to supersaturation and GBT. One aspect of these measurements, common to all levels of TGP of 1.12 Atms. and greater, was a decline in arterial p 0 2 with time. The implication of this decline is that the transport of oxygen to blood is being blocked by some mechanism, presumably at the gill level. Because many of the symptoms of GBT appear to depend on water TGP levels, they will be examined in more detail based on TGP. 5.4.2 GBT BETWEEN A TGP OF 1.1 AND 1.15 Atms.: In this range of TGP the overall response is that associated with hypoxia. That is, arterial blood pressure, heart rate and p 0 2 levels react in a manner typical of fish exposed to hypoxic water. Holeton and Randall (1967a,b) have shown that Rainbow trout exposed to water low in dissolved oxygen exhibit a decline in arterial p 0 2 . There is an associated rise in blood pressure and a fall in heart rate. There is a large increase in cardiac stroke volume to offset the decline in heart rate and allow maintenance of overall cardiac output. The increased blood pressure is thought to be the result of generalized vasoconstriction while the reduction in heart rate is thought to facilitate the synchronism between respiratory water flow and blood flow through the gills (Randall, 1982a). Except for stroke volume, which was not measured, all other cardiovascular characteristics associated with hypoxia were seen in fish exposed to TGP between 1.12and 1.15Atms. Blockage of Oxygen Transport: As described earlier, several factors indicate that bubble formation in the vascular system does not occur at TGP levels below 1.15 Atms. With the apparent absence of intravascular bubbles, the observed reduction in 132 arterial p 0 2 at lower levels of TGP must be caused by other factors. The most obvious, based on other symptoms, is the blockage of respiratory water flow by blisters in the lining of the mouth, blockage of flow by extracorporeal bubbles that appeared between the gill lamella or both of these effects acting in concert. If either or both of these effects are present, it should be expected that adjustments in ventilation will be made by the fish to offset the reduced transport of oxygen to blood. Holeton .and Randall (1967a, b) have shown that, over a wide range of hypoxic conditions, Rainbow trout increased ventilation volume dramatically so that net oxygen uptake does not decrease. If the blockage of respiratory water flow is the cause of declining arterial pG^, a study of the respiratory function during exposure to supersaturation would help confirm the blockage hypothesis. This question was addressed in the Phase II experiments and will be discussed in Section 5.7. Bubble Growth Thresholds: Since a response similar to that of hypoxia was observed at a TGP of 1.12 Atms., and no response occurred at 1.10 Atms., there appears to be a GBT threshold between these two levels of TGP. This is also the range of the lower threshold found in the database analysis, Section 4. Based on the earlier discussion of experimental results, this does not appear to be a threshold for bubble growth in the vascular system. However, it was clear the threshold at 1.10 to 1.12 Atms. corresponds to that for sub-dermal bubble growth in the skin and, in particular, the growth of tissue blisters in the buccal cavity. It was not established in these experiments that 1.10 to 1.12 Atms. TGP was also the threshold for the growth of extracorporeal bubbles observed in the water between gill lamella. It was concluded that, in order to determine this threshold, a detailed microscopic examination of the gills is required. This conclusion was also carried forward into the Phase II experimental studies. 133 Other Observations From the Literature: The external symptoms of GBT observed in these experiments agree with most observations from the literature (Nebeker et al, 1976; Rucker, 1975; Dawley and Ebel, 1975; Nebeker and Brett, 1976; Nebeker et al., 1980). One contrasting example is the data of Stroud and Nebeker (1976) for Steelhead trout exposed to a TGP of 1.1 Atms. Of the fish sampled at this TGP, the authors report hemorrhages in the gills in 17 out of 29 fish. Yet, microscopic examination of the gill revealed no intravascular bubbles. This is particularly unusual in that, at a TGP of 1.15 Atms., they report only one fish out of 29 had gill hemorrhages and there was still no evidence of intravascular bubbles. At a TGP of 1.20 Atms. they found only one fish out of 19 with gill hemorrhages; however, 18 of these fish showed extensive bubble formation in the gill arteries. The prevalence of hemorrhages at 1.10 Atms. is difficult to explain in terms of intravascular bubbles and implies other mechanisms were responsible for this damage. In the Phase I experiments, there was occasional evidence of gill hemorrhages; however, these were only at the highest TGP levels tested. In yet other data from the literature there is evidence that, in some environments, the lesions of external tissue caused by sub-dermal bubble growth can act as sites for fungal infections (Weitkamp, 1976). It has been speculated that these infections are a contributing factor in the death of fish (Weitkamp and Katz, 1980). In the Phase I experiments there were no indications of fungal infections in any of the fish, although some skin lesions were present. The presence of fungal infections will no doubt be highly dependent on water quality and general fish health. Therefore, the absence of such infections in these experiments cannot be taken as significant. Neither in the database analysis of Section 4 nor in other data from the literature is there any evidence that symptoms or mortality are dependent on water p02 for TGP levels below 1.15 Atms. (Stroud and Nebeker, 1976; Meekin and Turner, 1974; Nebeker et al., 1976; Rucker, 1975; Dawley and Ebel, 1975; Nebeker and Brett, 1976; Nebeker et al., 1980). This is consistent with the results of the Phase I experiments. p 0 2 levels varied from 100 to 250 mmHg. in these experiments; however, at any given TGP, there were no detectable differences in symptom severity or time to mortality that could be related to differences in water p 0 2 . 5.4.3 GBT BETWEEN A TGP OF 1.15 AND 1.25 Atms.: The response of the vascular system in fish exposed to these levels of supersaturation varied considerably; often with contrasting results. Fish No's. 6, 11, 17, and 27 all experienced major increases in arterial blood pressure in this range of TGP. Yet, Fish No. 15 and other fish tested showed no elevated blood pressure in this same TGP range. The only consistent results, above a TGP of 1.18 Atms., were declines in arterial p0 2 , elevated hematocrits and the presence of intravascular gill bubbles. Blockage of Oxygen Transport at the Gills: The decline of arterial p 0 2 above a TGP of 1.15 Atms. shows that fish continue to experience reduced oxygen transport to the blood. As suggested above, extracorporeal gill bubbles and blisters in the mouth may be the source of this problem. However, at the higher levels of TGP, blisters of tissue lining the mouth were either not well developed or absent entirely. Presumably, this was because the onset of lethal symptoms was more rapid at higher levels of TGP and buccal cavity blisters did not have time to form before death. Therefore, it is concluded that, at higher levels of TGP, intravascular bubbles, extracorporeal bubbles in the gill lamella or other mechanisms are responsible for the decline in p 0 2 . 135 It is not difficult to understand how blockage of respiratory water flow by extracorporeal bubbles would inhibit oxygen transport to the blood. However, the low arterial p 0 2 is a more complex problem to explain in terms of blockage of blood flow by intravascular bubbles. First, as pointed out in the theoretical section (Section 3), the gills are not the most likely sites for initial bubble formation. High vascular system pressures in this region would increase the threshold for bubble formation as reflected by the Ps term in Equation 4. The formation of bubbles in the gill vasculature would most likely occur just before death of the animal when blood pressure is falling. Figures 42 and 45 clearly show the drop in blood pressure that precedes death. As shown in the figures, this occurs only during the last few hours or minutes before death; whereas, the decline in arterial p 0 2 begins much earlier. The period over which intravascular bubbles existed in the gill vasculature was not examined in the Phase I experiments; however, this problem was addressed in the Phase II studies. Assuming that bubble formation in the gills does occur early, an explanation for low arterial p 0 2 can be developed around reduced blood transit time through the lamella. Because the heart is a form of positive displacement pump, flow continuity requires that blockage of flow in some gill arteries would increase flow velocity in the unblocked arteries. It follows that the time available for oxygen transport is reduced in the unblocked arteries. As a result, blood would not become fully oxygenated. An additional consideration is that under resting conditions, fish do not use all gill lamella for oxygen transport (Booth, 1978; Farrell, 1979 and Farrell, Daxboeck and Randall; 1979). That is, only about 2/3 of the available lamella are perfused with blood (Farrell, Daxboeck and Randall; 1979). The remaining lamella are recruited under conditions of hypoxia or swimming activity. Thus, it appears that fish have the ability 136 to withstand some blockage of gill blood flow before there is a decline in arterial p 0 2 . On the other hand, if extracorporeal gill bubbles have already induced hypoxia and full deployment of gill lamella, blockage by intravascular bubbles will have an immediate effect on oxygen transport. It should be remembered that a significant period is involved before a decline in arterial p 0 2 is observed. During the Phase I experiments, this time varied from a few hours, at high levels of TGP, to over 100 hours at a TGP level of 1.15 Atms. If bubbles do form early in the gill vasculature, there should be adequate time for their growth to lead to blockage of most of the gill vasculature. In this situation, it is expected that bubbles eventually impact oxygen transport. There remains however, the problem of the high blood pressure in the gills which would suggest other locations for initial bubble formation. The Phase II experiments produced additional insight into intravascular bubble formation in the gills and further discussion of this problem will be postponed until those results are examined. Other Blockage of the Cardiovascular System: There remains the possibility that bubbles form elsewhere in the cardiovascular system. As shown in the theoretical section, the most likely sites for initial bubble formation are the arterioles just upstream of tissue capillary beds. If this is the case, the explanation of declining arterial p 0 2 at high levels of TGP must lie entirely with the extracorporeal gill bubbles. Blockage of tissue and organ capillary beds can have dire consequences in terms of oxygen supply to these areas. However, in the absence of intravascular or extracorporeal bubbles in the gills, it should not effect p 0 2 at the dorsal aorta. An extreme case can be envisioned where generalized blockage of all tissue will stop return blood flow to the heart. In this case, oxygen in the dorsal aorta will decline as a result of utilization by red blood cells and vascular system tissue. However, it is difficult to see how the heart could maintain blood pressure or how the fish could live for the long periods during the gradual decline in arterial p02- Thus, in the absence of intravascular gill bubbles, one is left with extracorporeal bubbles blocking respiratory water flow as the most likely explanation of declining arterial pC^. Again, the Phase II experiments provide further information which help explain the low arterial pC^. Systemic Control Functions: The response of the vascular system to intravascular bubble formation appears to involve a conflict of systemic control functions in the animal. As described earlier, hypoxia caused by the blockage of oxygen transport elicits a general peripheral vasoconstriction, reduced heart rate and increased heart stroke volume. On the other hand, the response to rising blood pressure is one of peripheral vasodilation and a reduction of both heart rate and cardiac output (Randall, 1970). In either case, a reduction in heart rate will occur under conditions of hypoxia or elevated blood pressure. This was clearly the case in Fish 11 where heart rate was reduced up until attenuation of the pressure pulse made heart rate ambiguous (Figure 39). In Fish 6 which underwent the most rapid response to bubble formation, heart rate also became progressively slower up until the time of death. Blood Pressure Response to Bubble Growth: The increase in blood pressure observed in some fish exposed to supersaturation, along with the residual blood pressure at death, is interpreted as being caused by the volume of gas bubbles added to the vascular system. As shown in the theoretical section, the addition of gas to a closed system will cause the pressure to rise. In the cardiovascular systems of fish, as well as in other animals, systemic control of blood pressure will alter this 138 response somewhat. As pointed out above, the general response to an elevation in blood pressure is a dilation of peripheral circulation and a reduction in both heart rate and cardiac output. In addition, there can be an increase in blood flow to the kidneys which effectively reduces blood volume in other portions of the circulatory system (Eckert, 1983). There may also be reduced secretion of ADH from the pituitary gland which slows filtration rates in the kidney (Eckert, 1983). This also increases water loss which provides still another means of reducing blood volume. The kidney response is observed in many animals; however, it has not been confirmed for fish. Assuming a similar response in fish, it is conceivable that during the initial stages of bubble growth, systemic regulated adjustments to the vascular system could permit bubble growth without major changes in vascular system pressure. It would be anticipated there are limits in the ability of the system to withstand pressure increases due to the addition of bubble gas. The controlling factors would be the limiting balance between the rate of gas addition and the rate of water loss from the system. Certainly, if gas from growing bubbles is being added faster than systemic adjustments can be made, there will be a rise in pressure. In some environments, there may be other volume adjustments caused by a change in water flux at the gills. In freshwater fish, which are hyper-osmotic relative to their environment, there is a net uptake of water across the gills (Isaia, 1984). This is in the face of a hydrostatic pressure within the animal (i.e. blood pressure) greater than the hydrostatic pressure of the environment. For Rainbow trout, the inward water flux is driven by a colloid osmotic pressure gradient of about 350 mosm. (Holmes and Donaldson, 1969). Because of this high osmotic pressure, there is little chance of a reversal of water flux at the gills of freshwater fish caused by elevated blood pressure. However, an elevated blood pressure will slow this influx somewhat. It will be recalled 139 that in some experimental fish, there was a doubling or near doubling of blood pressure caused by bubble growth. For marines fishes, blood plasma is either hypo-osmotic or nearly iso-osmotic relative to sea water. In the first case, there is a net efflux of water across the gills. An elevated blood pressure resulting from intravascular bubbles will cause an increase in this efflux. Fish that are iso-osmotic relative to the environment may begin loosing water across the gills as a result of bubble growth. In either case, increased water loss by the kidneys or across the gills should produce a concentration of red blood cells and other blood components as gas replaces water in the system. An additional mechanism for water loss is the movement of intravascular water into tissue intercellular spaces. This water flux would be driven by the elevated blood pressure observed at the higher levels of TGP. In this case, opposing colloid osmotic gradients would be considerably less than those involved in the movement of water across the gills. Again, the net effect should be reflected as an increase in hematocrit. Hematocrit: Hematocrit can also increase as a result of other physiological processes. Under conditions of hypoxia, acid base disturbances have been shown to induce swelling of red blood cells with corresponding increases in hematocrit (Baroin et al., 1984; Nikinmaa, 1982; Nikinmaa and Huestis, 1984; Heming et al., 1987 and Tufts, 1988). It has been suggested that during hypoxia, recruitment of red cells from the spleen and kidney can serve to increase oxygen carrying capacity of the blood (Ostroumova, 1964; Stevens, 1968; Johansen and Hansen, 1967 and Yamamoto, 1980). Such recruitment will also be reflected as an increase in hematocrit. In the Phase I experiments, increases in hematocrit were clearly present 140 at TGP levels above 1.18 Atms. In fact, hematocrit fractions were found to increase by as much as 85% at the higher levels of TGP. It is unclear as to whether these increases are the result of water displacement, the swelling of red blood cells, red cell recruitment or combinations of all three. However, the answer to this question may lie in observations from the literature. Weber (1982); Baroin et al., (1984a,b), Nikinmaa and Huestis (1984), Heming et al. (1987) and Tufts (1988) have shown that Rainbow trout red blood cells (RBC) swell as part of a defense against intracellular acid-base disturbances. A decline in plasma pH will seriously disrupt the oxygen carrying capacity of Root - Bohr sensitive hemoglobins which are characteristic of Rainbow trout (Riggs, 1979 and Foster and Steen, 1969). This is particularly true for extended exposure to deep hypoxia. That is, a buildup of lactic acid produced by anaerobic metabolism can lead to a metabolic acidosis and reduced blood pH (Holeton and Randall, 1967a,b and Thomas and Hughes, 1982). In pure hypoxia, which is usually accompanied by hyperventilation, there is seldom a problem of CO2 accumulation in the blood (Holeton and Randall, 1967a,b and Thomas and Hughes, 1982). An accumulation of CO2 will decrease blood pH (Ferguson & Black, 1941; Cameron, 1971; Cameron and Randall, 1972 and Eddy et al., 1977). This, combined with any metabolic acidosis, will reduce blood oxygen carrying capacity even further. In the case of hypoxia produced by the blockage of respiratory pathways, it can be expected that, as oxygen delivery to the blood is impeded, CO2 removal from the blood will be restricted. Thus, CO2 buildup can compound oxygen uptake by reducing blood pH even further. In fish, the problem of blockage can become even more acute since they also use respiratory pathways for removal of metabolites and osmotic regulation. In many ways, the situation is similar to that examined by Holeton and Randall (1967a,b) where Rainbow 141 trout were subjected to hypoxic water in a closed swimtube respirometer. In this environment, hypoxia was induced through utilization of oxygen by the fish while C02 and metabolic wastes in the water increased as a result of respiration and other excretory processes. This should have caused a gradual reduction of total C02 excretion at the gill and compounded the acidosis created by an observed buildup of lactate. Holeton and Randall did observe an increase in blood hematocrit accompanied by red blood cell swelling. However, no evidence could be found for increases in red cell count. Although the authors noted increases in hematocrit caused by exposure to the combined hypoxia/hypercapnia environment, the actual changes are not reported. Also, the time course over which the hypoxia took place was not described. Based on these results it appears that red blood cell swelling will take place in situations involving both metabolic and respiratory acidosis. In other experiments, involving Rainbow trout exposed to hypoxia, Thomas and Hughes (1982) found hematocrit rose quickly from 0.20 to 0.23 during 24 hours of exposure to a water p 0 2 of 60 mmHg. However, in these experiments it was not determine if red blood swelling occurred or if RBC recruitment was present. They did find that blood pH reflected an initial state of respiratory alkalosis which they attributed to hyperventilation. Over a 24 hour period, blood pH gradually returned to normal. In yet other experiments with Rainbow trout exposed to deep hypoxia (water p 0 2 of 40 mmHg), Thomas and Hughes (1982) found an initial metabolic acidosis of the blood brought on by lactate buildup. This was followed by a recovery of pH and then a reversal to an alkalotic state. Again a very rapid increase in hematocrit from 0.23 to 0.29 was noted. If it is assumed that the increases in hematocrit observed by Thomas and Hughes are the result of red blood cell swelling, then the maximum change in hematocrit produced by this effect was a 27% increase. Even if a 142 component of this was due to RBC recruitment, the maximum change would still be 27%. Fish in the Phase I experiments exposed to high levels of TGP showed increases in hematocrit of as much as 85%. Thus, it appears that a significant portion of the observed hematocrit increase is due to other causes such as the displacement of intravascular water in the vascular system by bubble gas. 5.5 P H A S E I C O N C L U S I O N S : The Phase I experimental studies produced an improved understanding of the physiological response in fish exposed to supersaturated water. At water TGP above 1.1 Atms., the dominant response is one of hypoxia. That is, there is clear evidence that oxygen transport to the blood is being impeded somewhere in the transport pathway. The indications are that between a TGP of 1.1 and 1.15 Atms., the blockage is due to extracorporeal bubbles forming between gill lamella, the formation of blisters in the buccal cavity or a combination of these two. Above a TGP of 1.15, where buccal cavity blisters are less frequent or non existent, intravascular bubbles within the gill lamella are common. These bubbles along with the extracorporeal gill bubbles may be the blockage mechanism at the higher levels of TGP. These experiments demonstrate that a TGP threshold exists between 1.10 and 1.12 Atms. for the growth of sub-dermal bubbles on the external body surfaces and lining of the mouth. This corresponds to the lower threshold for mortality derived from the database analysis. However, it is not known that buccal cavity blisters are the cause of mortality. This is also the TGP range of thresholds for extracorporeal gill bubble growth. However, this threshold could not be established from the Phase I experimental data. Based on the presence of intravascular bubbles in the gills, 143 elevated hematocrit and residual blood pressures following death, it appears that a threshold for intravascular bubble growth exists in the TGP range of 1.15 to 1.25 Atms. Above a TGP of 1.25 Atms., there is clear evidence that blood pressure reflects the presence of growing bubbles in the vascular system. However, between a TGP of 1.15 and 1.25 Atms., the response of blood pressure before death was highly variable. As a result, it was a poor indicator of bubble growth thresholds for the vascular system. The most reliable indicators of intravascular bubble growth were hematocrit and visual evidence of intravascular gill bubbles. Microscopic examination of gill lamella gave the clearest confirmation of the presence of both intravascular and extracorporeal gill bubbles. From the Phase I studies it was clear that additional experiments were required before intravascular and extracorporeal bubble growth thresholds can be defined. In addition, there remained the task of correlating these thresholds with mortality and the thresholds indicated in the literature database analysis. 5.6 PHASE II EXPERIMENTAL RESULTS: 144 The Phase II experiments expanded on the Phase I results and provided clearer indications of the various bubble growth thresholds associated with GBT. In addition, the experiments focused on specific physiological responses in fish as a means of defining the relationship between bubble growth and mortality. The results of the Phase II experiments will be presented in the order of the individual experimental series. 5.6.1 SERIES 1 THROUGH 5 EXPERIMENTS: The Series 1 through 5 experiments involved exposing groups of 12 fish (6 cannulated and 6 un-cannulated) to various levels of water TGP and p 0 2 . External symptoms, internal and external conditions of the gills and blood parameters were surveyed during the experiments. The results are summarized in a series of figures which will be examined next. The individual series will be described in the order of increasing water TGP. Table II lists the abbreviations used in the various bar charts that will be presented. In the results, Fish No's 1 through 6 always correspond to the cannulated fish while Fish No's 7 through 12 are the un-cannulated fish. TABLE II: ABBREVIATIONS FOR SEVERITY OF SYMPTOMS CHARTS LAMELLA - Extracorporeal bubbles between gill lamella. VASCULAR - Intravascular bubbles in gill lamella. OPERCULA - Sub-dermal bubbles on opercular flaps. FINS - Sub-dermal bubbles in fin rays. BUCCAL - Sub-dermal bubbles in the lining of the buccal cavity. 145 In general, it was found that cannulated fish did not live as long as un-cannulated fish exposed to supersaturated water. Figure 46 summarizes these findings in terms of time to mortality versus water TGP for the two groups. It will be noted that the times to mortality in the figure do not vary greatly in the TGP range of 1.10 through 1.17 Atms. However, at a TGP of 1.19 Atms., time to mortality drops to roughly one half or one third of the time required at lower TGP. Both cannulated and un-cannulated fish showed similar signs of GBT throughout the experiments. However, it was impossible to determine if decreased survival of the cannulated fish was due to the stress of cannulation, increased susceptibility to bubble growth caused by handling and surgery, or the presence of the cannula. Series 1 (Water TGP = 1.10 Atms.. pOow = 170 mmHg.): In this series of experiments two fish showed external signs of GBT. Of these, one fish (Fish 4) died early and exhibited a decline in arterial p 0 2 which suggested a blockage of oxygen transport as was observed in the Phase I experiments. Furthermore, this fish was the only one to show a significant number of extracorporeal bubbles between gill lamella. Other blood parameters were at normal levels. Although there was a decline in arterial p 0 2 at the last measurement, it was not low enough to suggest acute hypoxia. In Figure 47 the variations in arterial p 0 2 for the six cannulated fish are shown as a function of time of exposure. Except for Fish 4, only small changes occurred during the experiment. Although three fish died at various times during the experiment, Fish 4 was the only one to show signs of GBT that would be considered lethal. Fish 8 also showed bubble growth between gill lamella. However, there was no evidence of hypoxia and the animal lived without signs of stress for the full 362 hours of the experiment. T I M E T O D E A T H A S A F U N C T I O N O F W A T E R T G P S E R I E S 1, 2, 3 , 4 , & 5 146 4 0 0 T I M E T O D E A T H - H r s . 3 0 0 -2 0 0 1 0 0 1.1 1.15 1.2 W A T E R T O T A L G A S P R E S S U R E - A t m s . 1 . 2 5 • CANNULATED FISH * + UN-CANNULATED FISH 12 FISH IN EACH TREATMENT 6 CANNULATED AND 6 UN-CANULLATED FISH FIGURE 46: Time to Death as a Function of Water TGP. ARTERIAL BLOOD p02 VARIATION WITH TIME EXPERIMENTAL SERIES 1 WATER TGP • 1.10 Atms., p02w • 170 mmHg. 147 ARTERIAL p02 - mmHg. 140 -1 0 6 56 125 198 265 317 362 400 TIME OF EXPOSURE - HRS. • FISH 1 + FISH 2 * FISH 3 • FISH 4 X FlSH 5 0 FISH 6 FIGURE 47: Arterial p 0 2 Variation With Time, Experimental Series 1. 148 In Figure 48, the variations in arterial blood pH and hematocrit are shown for the cannulated fish as a function of exposure time. It is clear that little change in either occurred over the 362 hours of exposure. Figure 49 shows the level of severity of various symptoms at death in the 12 animals tested. The scale for these bar charts, and all others to follow, corresponds to the scaling system described in the Experimental Methods Section above. In this experimental series most animals were alive at the end of the experiment and were killed to make the necessary measurements. This procedure was followed in each of the series when fish remained alive through the end of the experiment. Series 3 (Water TGP = 1.12 Atms.. pOow = 175 mmHg.'): The results of these experiments showed several fish with declines in arterial p 0 2 that suggest a blockage of oxygen transport. In Figure 50, arterial p 0 2 is shown for the six cannulated fish as a function of exposure time. Except for Fish 1 and Fish 6, major reductions in arterial p 0 2 occurred. Furthermore, GBT related mortality occurred in all but three of the cannulated fish and in one of the un-cannulated fish (Figure 46). In addition to declines in arterial p 0 2 , there were corresponding declines in arterial blood pH as shown in Figure 51. The decreases in pH correlate with the p 0 2 declines of Figure 50. However, arterial hematocrit showed little change in any of the cannulated fish. This observation is consistent with the results of the Phase I experiments at low levels of TGP. In Figure 52, the severity of symptoms at death are shown for the six cannulated fish in the upper plot, and for the six un-cannulated fish in the lower plot. It is clear that in all cases there were external blisters on the opercula and fins, but the degree varied considerably from fish to fish. ARTERIAL HEMATOCRIT VARIATION WITH TIME EXPERIMENTAL SERIES 1 WATER TGP • 1.10 Atms., p02w - 170 mmHg. 149 0.60 ARTERIAL HEMATOCRIT FRACTION 0.00 66 126 198 TIME OF EXPOSURE 265 HRS. 400 FISH 1 a FI8H 4 FI8H 2 FISH 6 * FI8H 3 « FISH 6 ARTERIAL BLOOD pH VARIATION WITH TIME EXPERIMENTAL SERIES 1 WATER TGP - 1.10 Atms.. p02w - 170 mmHg. ARTERIAL pH - mmHg. 8.10 8.00 7.90 7.80 7.70 7.60 7.60 -* 4-* 4-* i * i * + * * 9 X X • X X X X • _ o 0 0 0 0 0 -0 0 -I 1 1 1 1 i 1 -1 0 6 66 126 198 266 317 362 400 TIME OF EXPOSURE - HRS. FISH 1 + FISH 2 * FI8H 3 ° FISH 4 x FI8H S 0 FI8H 6 FIGURE 48: Arterial Hematocrit and pH Variation with Time, Series 1. 8EVERITY OF 8YMPTOMS AT DEATH WATER TGP - 110 Atme., p02w • 170 mmHg. EXPERIMENTAL 8ERIES 1 8YMPTOM 8EVERITY: 0 - 3 3 r 1 j r LAMELLA VASCULAR EH3 OPERCULA • S FIN8 BUCCAL 3 4 FI8H NUMBER 6 8EVERITY OF 8YMPTOMS AT DEATH WATER TOP - 110 Atms.. p02w - 170 mmHg. 8YMPTOM 8ERERITY: 0 - 3 31 ! ! r LAMELLA VA8CULAR m OPERCULA ftV FIN8 B BUCCAL 8 0 10 FI8H NUMBER ti 12 FIGURE 49: Severity of Symptoms at Death. Experimental Series l ARTERIAL BLOOD p02 VARIATION WITH TIME 1 5 1 EXPERIMENTAL SERIES 3 150 50 WATER TGP • 1.12 Atms. ,p02w • 175 mmHg. ARTERIAL p02 - mmHg. $ o 1 ^ 0 • 0 • $ ; • 0 + _ * * X • • X 1 i i -1 0 6 56 127 198 249 296 367 400 T I M E O F E X P O S U R E - H R S . • FISH 1 + FISH 2 * FISH 4 ° FISH 4 x FlSH 6 0 FISH 6 FIGURE 50: Arterial Blood p02 Variation with Time, Experimental Series 3. ARTERIAL HEMATOCRIT VARIATION WITH TIME EXPERIMENTAL 8ERIES 3 WATER TGP • 112 Atms., pQ2w • 175 mmHg. 152 ARTERIAL HEMATOCRIT FRACTION 0.60 0.00 1 0 6 66 127 198 249 296 367 400 TIME OF EXPOSURE - HRS. FI8H 1 + FI8H 2 * FI8H 3 n FI8H 4 * FI8H 6 » FI8H 8 ARTERIAL BLOOD pH VARIATION WITH TIME EXPERIMENTAL SERIES 3 WATER TGP - 112 Atms., p02w - 176 mmHg. ARTERIAL pH - mmHg. 8.10 8.00 + + * * + a . 7.90 H X 0 9 7.80 -* X + 7.70 a + 7.60 - * x • • 7.60 • < 1 1 l -1 0 6 66 127 198 249 296 367 400 TIME OF EXPOSURE - HRS. FI8H 1 + FISH 2 * FI8H 3 a FISH 4 x F»H 6 o FISH 6 FIGURE 51: Arterial Hematocrit and pH Variation with Time, Series 3. SEVERITY OF SYMPTOMS AT DEATH WATER TGP - 1.12 Atms., p02w - 176 mmHg EXPERIMENTAL SERIES 3 1 5 3 SYMPTOM SEVERITY: 0 - 3 1 2 3 4 6 6 FISH NUMBER SEVERITY OF SYMPTOMS AT DEATH WATER TGP - 1.12 Atms.. p02w - 175 mmHg. SYMPTOM SEVERITY: 0 8 LAMELLA VASCULAR l i l OPERCULA Wffl FINS [H! BUCCAL 9 10 FISH NUMBER 11 FIGURE 52: Severity of Symptoms at Death, Series 3. 154 Also, except for Fish No. 1, there were varying degrees of extracorporeal bubble growth between gill lamella accompanied by blisters in the buccal cavity. Figure 53 is a photograph showing an example of the extracorporeal bubbles that were usually present. The bubbles shown in the photograph are typical of those cases assigned a number of one for the level of severity. The bubbles appear to be randomly distributed throughout the primary and secondary lamella. The ones between secondary lamella were quite stable and were difficult to dislodge with a needle. There was no evidence of intravascular bubbles in these gill lamella. The same was true for all fish examined in this series. Figure 54 is a photograph showing large blisters on the upper part of the mouth of one of the experimental animals tested at a TGP of 1.12 Atms. Figure 55, taken of the same animal but at a slightly different angle, shows blisters lining the entrances to the gills. These blisters were of a size and number that would suggest interference with water flow to the gills. In this fish, the severity of buccal cavity blisters was assigned a value of 3. Figures 56 and 57 show examples of blisters (severity level between 2 and 3) that were found on the opercular flaps of fish exposed to the lower levels of TGP. Examples of the blisters occurring between fin rays are shown in Figures 58 and 59. Figure 58 shows a segment of the dorsal fin from one fish while Figure 59 shows the caudal fin of the same animal. In both cases, the level of severity is 2. If one examines the array of symptoms and their severity (Figure 52), it is evident that a correlation exists between the overall severity of symptoms and the time to mortality. It will be noted that in the first six hours of the experiment, arterial p 0 2 is elevated relative to the pre-exposure levels. This reflects the higher levels of dissolved oxygen in supersaturated water. This observation was common to all experiments where experimental water p 0 2 was above that of the pre-exposure holding water. FIGURE 53: FIGURE 53: Extracorporeal Bubbles in Gills. 156 FIGURE 55: Subdermal Bubbles in Mouth. FIGURES 56 AND 57: 157 FIGURE 57: Opercular Bubbles. FIGURES 58 AND 59: 159 Series 4 (Water TGP = 1.15 Atms.. pQow = 183 mmHg.): The results of this experimental series show that all cannulated fish had varying rates of declining arterial pO*2 (Figure 60). At the time of measurement, none of the levels were considered acute. However, death usually followed the lowest measured value. All but one of the cannulated fish and one of the un-cannulated fish died during the experiment. All fish showed signs of GBT and, as in experimental Series 3, the time to mortality could be correlated with the severity of symptoms. All cannulated fish showed a decline in arterial pH (Figure 61), which corresponded closely to the declining p 0 2 . Extracorporeal bubbles were present to varying degrees throughout the gills of all fish. Except for Fish No. 3, blood hematocrit showed little variation for the cannulated fish. In Fish No. 3, there was a 67% increase in hematocrit over the course of the experiment. Furthermore, this fish, Fish 4 and Fish 10 were the first to show signs of intravascular bubble growth in the gill arteries (Figure 62). In fish 4 and fish 10, bubble growth was at a minimum level and the times to mortality were not much different from those for fish without vascular bubbles. Figures 63 and 64 are a collection of microscope photographs taken of gill lamella from some of these fish and from fish in series at higher levels of TGP. In all photographs vascular bubbles are clearly present in the filamentary arteries of the primary lamella. Of all the gill lamella examined microscopically, only one fish, exposed to a water TGP of 1.19 Atms.. showed the presence of bubbles in the secondary lamella. These bubbles had clearly grown into the region from the filamentary arteries of the primary lamella. With few exceptions, bubbles appear to start at the distal ends of the filamentary arteries and grow toward the base of the primary lamella. ARTERIAL BLOOD p02 VARIATION WITH TIME EXPERIMENTAL SERIES 4 WATER TGP - 1.15 Atms., p02w - 183 mmHg. 160 ARTERIAL p02 - mmHg. 160 20 h -1 0 6 53 132 201 245 301 345 400 TIME OF EXPOSURE - HRS. • FISH 1 + FISH 2 * FISH 3 • FISH 4 x FISH 5 0 FISH 6 FIGURE 60: Arterial Blood pC>2 Variation with Time, Series 4. ARTERIAL HEMATOCRIT VARIATION WITH TIME EXPERIMENTAL SERIES 4 WATER TGP • 1.16 Atms.. p02w - 183 mmHg. ARTERIAL HEMATOCRIT FRACTION 0.50 0.00 63 132 201 245 301 345 400 TIME OF EXPOSURE - HRS. • FI8H 1 + FI8H 2 * FI8H 3 D FI8H 4 * FI8H 6 » FI8H 6 ARTERIAL pH VARIATION WITH TIME EXPERIMENTAL SERIE8 4 WATER TGP - 1.16 Atms., p02w - 183 mmHg. ARTERIAL pH 8.10 6.00 7.90 7.80 7.70 7.60 7.60 - 4-i - ' 8 • * a * •i-+ 8 X ft X R * 4-X a « t 0 i < X I 1 1 0 6 63 132 201 245 301 346 400 TIME OF EXPOSURE - HRS. • FI8H 1 + FISH 2 * FI8H 3 ° FISH 4 * FISH 6 « FI8H 6 FIGURE 61: Arterial Hematocrit and pH Variation with Time, Series 4. SEVERITY OF SYMPTOMS AT DEATH WATER TGP - 1.16 Atme., p02w • 183 mmHg. EXPERIMENTAL SERIES 4 162 SYMPTOM SEVERITY: 0 LAMELLA VASCULAR E H OPERCULA MM FINS (=3 BUCCAL 3 4 FISH NUMBER SEVERITY OF SYMPTOMS AT DEATH WATER TGP - 1.16 Atma., p02w - 183 mmHg. SYMPTOM SEVERITY: 0 - 3 9 10 FISH NUMBER LAMELLA VASCULAR 1 1 OPERCULA Wffl FINS BUCCAL FIGURE 62: Severity of Symptoms at Death, Series 4. 164 FIGURE 64: a, b, c & d: Vascular Bubbles in Gills. Series 2 Mater TGP = 1.17 Atms.. pQ 2w = 195 mmHg.): In the Series 2 experiments every fish showed varying degrees of intravascular bubble growth in gill lamella (Figure 67). Increases in hematocrit were seen in each of the cannulated fish (Figure 66). Arterial blood pH and p 0 2 dropped dramatically (Figures 66 and 65, respectively) and all fish died during the experiment (Figure 46). Interestingly, the external signs of GBT, such as blistering of tissue, were not as pronounced as in the experiments at lower levels of TGP. Presumably, this is because fish died from other causes before these symptoms could become well developed. Also, it was clear that the number of extracorporeal bubbles were fewer than at lower levels of TGP. On the other hand, the bubbles that were present appeared to be larger. As with previous experimental Series, a rise in p 0 2 during initial stages of exposure was seen in many of the fish. This again reflects the elevated p 0 2 levels in the water. As exposure time increased, this initial rise in p 0 2 was offset by the apparent blockage of oxygen transport to blood. Series 5 (Water TGP = 1.19 Atms.. pOow = 201 mmHg.): At a TGP of 1.19 Atms. the time of survival of all fish declined dramatically (Figure 46). Arterial p 0 2 declined rapidly for all cannulated fish (Figure 68) and presumably for the un-cannulated fish as well. Also, there was a high level of intravascular bubble formation in the gills of all fish (Figure 70). As with previous experiments, arterial pH dropped and hematocrit rose, but more rapidly than in any of the other experiments (Figure 69). The trend in decreasing numbers of extracorporeal bubbles in the gills continued in this experimental series. Again, the sizes of the bubbles were larger than those in previous series. Also, very few external blisters were present in these animals, presumably because of the short time of survival. 1 ARTERIAL BLOOD p02 VARIATION WITH TIME EXPERIMENTAL SERIES 2 WATER TGP • 1.17 Atms., p02w • 195 mmHg. 160 ARTERIAL p02 - mmHg. X X 140 - * * • 120 - 1 X 100 + • 80 0 • * X 60 0 + * 40 X 20 • i 1 -1 0 6 53 133 210 240 300 TIME OF EXPOSURE - HRS. • FISH 1 + FISH 2 * FISH 3 • FISH 4 x FISH 5 0 FISH 6 FIGURE 65: Arterial Blood pC>2 Variation with Time, Series 2. ARTERIAL HEMATOCRIT VARIATION WITH TIME EXPERIMENTAL SERIES 2 WATER TGP • 1.17 Atms.. pQ2w - 196 mmHg. 167 ARTERIAL HEMATOCRIT FRACTION 0.60 -1 0 6 63 133 210 240 300 TIME OF EXPOSURE - HRS. FI8H 1 + FI8H 2 * FISH 3 ° FI8H 4 * FISH 6 ° FI8H 6 ARTERIAL BLOOD pH VARIATION WITH TIME EXPERIMENTAL SERIE8 2 WATER TGP - 117 Atms., p02w • 196 mmHg. ARTERIAL pH - mmHg. 8.10 6.00 7.90 * 7.80 X + i 0 * 7.70 - X X 7.60 - * • 0 7.60 + • 7.40 J , » -1 0 6 63 133 210 240 300 TIME OF EXPOSURE - HRS. FI8H 1 + FI8H 2 * FI8H 3 a FI8H 4 * FISH 6 <> FISH 6 FIGURE 66: Arterial Hematocrit and pH Variation with Time, Series 2. SEVERITY OF SYMPTOMS AT DEATH WATER TGP - 1.17 Atms., p02w - 196 mmHg EXPERIMENTAL SERIES 2 168 SYMPTOM SEVERITY: 0 - 3 FISH NUMBER SEVERITY OF SYMPTOMS AT DEATH WATER TGP • 1.17 Atms., p02w - 196 mmHg. SYMPTOM SEVERITY: 0 - 3 7 8 9 10 11 12 FISH NUMBER FIGURE 67: Severity of Symptoms at Death, Series 2. ARTERIAL BLOOD p02 VARIATION WITH TIME EXPERIMENTAL SERIES 5 150 50 WATER TGP • 1.19 Atms., p02w • 201 mmHg. ARTERIAL p02 - mmHg. + + • i • X 0 + 0 * X + 0 • I I I 1 * i -1 0 6 23 56 80 100 TIME OF EXPOSURE - Hrs. • FISH 1 + FISH 2 * FISH 3 • FISH 4 x FlSH 6 0 FISH 6 FIGURE 68: Arterial Blood p02 Variation with Time, Series 5. ARTERIAL HEMATOCRIT VARIATION WITH TIME EXPERIMENTAL SERIES 6 WATER TGP - 119 Atms., p02w - 201 mmHg. 170 ARTERIAL HEMATOCRIT FRACTION 0.6 6 23 66 80 100 TIME OF EXPOSURE - Hrs. FI8H 1 + FISH 2 * FI8H 3 ° FI8H 4 * FISH 6 * FI8H 8 ARTERIAL BLOOD pH VARIATION WITH TIME EXPERIMENTAL SERIES 6 WATER TGP - 119 Atms., p02w • 201 mmHg. 8.1 ARTERIAL BLOOD pH 6 -7.9 * * o • * 7.8 B + 7.7 * + + 7.6 -7.6 a X 0 * 7.4 1 1 • -1 0 6 23 66 80 100 TIME OF EXPOSURE - Hrs. FI8H 1 + FI8H 2 * FISH 3 a FISH 4 x FI8H 6 * FISH 6 FIGURE 69: Arterial Hematocrit and pH Variation with Time, Series 5. 8EVERITY OF 8YMPTOM8 AT DEATH WATER TQP • 119 Atme., p02w - 201 mmHg. EXPERIMENTAL 8ERIES 6 171 SYMPTOM SEVERITY: 0 - 3 1 2 3 4 6 6 FISH NUMBER 8EVERITY OF SYMPTOMS AT DEATH WATER TGP - 119 Atms., p02w - 201 mmHg SYMPTOM SEVERITY: 0 - 3 7 6 9 10 11 12 FISH NUMBER FIGURE 70: Severity of Symptoms at Death, Series 5. 172 5.6.2 Series 6 (Water TGP = 1.19 Atms.. pOow = 201 mmHg.): In this series of experiments the levels of adrenaline and noradrenaline were measured in six cannulated fish during exposure to supersaturated water. Figure 71 shows the variation in adrenaline for the six animals as a function of exposure time. In this case, Fish No's. 1, 3, 4 and 5 all showed increasing levels of adrenaline as exposure time increased. Figure 72 shows the variation in noradrenaline with time for the six fish. Only Fish 3 exhibited a consistent increase in noradrenaline with exposure time. 5.6.3 Series 7 (Water TGP = 1.19 Atms.. pOow = 201 mmHg.): In this experimental series the ventilation performance of three fish was monitored using the van Dam respiratory chambers described in the methods section. Figure 73 shows the variation in both ventilation volume and frequency as a function of the time of exposure. Only one fish lived the full 80 hours of exposure. However, it was clear that all fish showed increases in both ventilation volume and frequency as the exposure period lengthened. In Fish 2, which lived the longest, a decline in both ventilation volume and frequency was measured 20 minutes before the fish died. 5.6.4 Series A. B. C. and Series 4 (Water TGP = 1.15 Atms.. pOow = 100. 125 and  183 mmHg.): In these experiments, water TGP was held constant while water p 0 2 was varied. Times to mortality were monitored along with the condition of gills. Twelve fish, Series A and B, were tested at a p 0 2 of 100 mmHg. Experimental Series C involved six fish exposed at a p 0 2 of 125 mmHg. The Series 4 data are the same as that for the un-cannulated fish described in Series 4 earlier in this section. The results of these experiments (Figure 74) clearly show there is strong dependence of time to mortality on water p 0 2 . Furthermore, a significant transition occurs between a p 0 2 of 100 and 125 mmhg. ADRENALINE VARIATION WITH TIME TGP - 1.19 Atms. p02 - 201 mmHg. SERIES 6 ADRENALINE - Pico gr./mL. 25 20 15 10 5 - * -• + X X -0 0 • • * 5 • 0 $ I I X. -10 0 10 20 30 40 50 60 70 80 TIME OF EXPOSURE - Hrs. • F I S H 1 + F I S H 2 * F I S H 3 • F I S H 4 x F I S H 5 0 F I S H 6 FIGURE 71: Adrenaline Variation with Time, Experimental Series 6. NORADRENALINE VARIATION WITH TIME TGP - 1.19 Atms. p02 • 201 mmHg. SERIES 6 174 NORADRENALINE - Pico gr./mL. 6 4 2 -X * - 0 0 • + ft 1 1 * i i • X 0 X -10 0 10 20 30 40 50 60 70 80 TIME OF E X P O S U R E - Hrs. • F I S H 1 + F I S H 2 * F I S H 3 O F I S H 4 x F I S H 5 0 F I S H 6 FIGURE 72: Noradrenaline Variation with Time, Experimental Series 6. VENTILATION VOLUME VERSUS TIME EXPERIMENTAL SERIES 7 WATER TGP • 119 Atms.. p02w • 201 mmHg. 175 VENTILATION VOLUME - Ml./Min. 250 200 160 100 60 20 40 60 80 TIME OF EXPOSURE - Hrs. D FI8H 1 + FI8H 2 * FI8H 3 120 100 80 60 40 20 VENTILATION FREQUENCY VERSUS TIME EXPERIMENTAL SERIES 7 WATER TGP - 119 Atms., p02w - 201 mmHg. VENTILATION FREQUENCY - Breathe/Mln. ft + 20 40 60 TIME OF E*XP08URE - Hrs. 80 ° FI8H 1 + FI8H2 * FI8H 3 NOTE: All fish hsd vascular systsm bubblss at dssth. FIGURE 73: Ventilation Volume and Frequency Versus Time, Series 7. 176 As described earlier, this is similar to the results obtained with Coho salmon by Rucker (1975) at a water TGP of 1.19 Atms. In that case, the transition in time to mortality occurred at a water p 0 2 of 249 - 275 mmHg. The conclusion drawn was that the strong transition in time to mortality represents a threshold for the cause of mortality. Presumably this was due to bubble growth. In Table III the presence or absence of intravascular gill bubbles at death is shown for the Phase II experimental series A, B, C and 4. These data also confirm that the transition in time to mortality correlates well with the presence of intravascular gill bubbles. TABLE III: SURVEY OF INTRAVASCULAR GILL BUBBLES + indicates bubbles were present. - indicates bubbles were not present. p 0 2 - in mmHg. FISH NO. BUBBLES? pOp FISH NO. BUBBLES? pOo A1 + 100 C1 125 A2 + 100 C2 - 125 A3 + 100 C3 - 125 A4 + 100 C4 + 125 A5 - 100 C5 - 125 A6 + 100 C6 - 125 B1 - 100 4-7 - 183 B2 + 100 4-8 - 183 B3 + 100 4-9 - 183 B4 + 100 4-10 + 183 B5 + 100 4-11 - 183 B6 + 100 4-12 - 183 TIME TO MORTALITY VERSUS WATER p02 1 7 7 FOR CONSTANT TOTAL GAS PRESSURE TGP - 1.15 Atms. TIME TO MORTALITY - Hrs. 400 300 200 100 100 150 200 WATER p02 Series A + Series C * Ser ies 4 D Ser ies B Note: One fish in Series 4 did not die in 400 hours. FIGURE 74: Time to Mortality Versus Water p 0 2 for Constant TGP. 178 5.6.5 OTHER RESULTS: In addition to the results described above, several supplemental studies were performed during the Phase II experiments. For example, tissue samples were periodically taken from muscle, liver and heart for microscopic examination. When intravascular bubbles were found in the gill lamella, intravascular bubbles were frequently found in the other tissue samples as well. Although bubbles were found less frequently in heart and liver tissue, they were again of intravascular origin. During experiments at a water TGP of 1.19 Atms., three fish, not included in those described above, were prematurely killed when their blood showed an initial rise in hematocrit and before the development of external stress symptoms (i.e. violent swimming). In each case, intravascular bubbles had started to grow in the gill lamella. The degree of bubble development, however, was not as severe as seen in other fish at the same TGP just before death. This observation, combined with the blood pressure response of the Phase I experiments, imply that intravascular bubble growth in the gill lamella begins early and is not the result of declines in blood pressure just before death. Similarly, two fish exposed to a water TGP of 1.12 Atms. were killed after 24 hours of exposure. This was done to determine if extracorporeal bubbles were present in the gills before declines in p 0 2 occurred or the development of severe sub-dermal bubble growth in the mouth. In both fish it was clear that extracorporeal bubbles were present between gill lamella. Again, as with intravascular bubbles, the degree of development was not as severe as that seen much later in the exposure period. 5.7 DISCUSSION OF PHASE II RESULTS 179 The results of these experiments confirm the observations of the Phase I experimental studies and refine the definition of bubble growth thresholds. In addition, significant information was added to the understanding of bubble growth and the physiological response of fish to this growth. As with the Phase I experiments, fish that experienced severe forms of sub-dermal bubble growth on the external skin also showed signs of minor hemorrhaging from lesions produced by these bubbles. However, the lesions were not considered to be severe enough, compared to other symptoms, as to contribute significantly to death of any of the animals. 5.7.1 EXTRACORPOREAL AND SUB-DERMAL BUBBLE GROWTH THRESHOLDS: Although fish of the Phase I experiments showed no response to supersaturation at a water TGP of 1.10 Atms., the Phase II experimental animals did show a limited response at 1.10 Atms. This difference between the two experimental Phases was most likely due to the number of fish examined. It was clear in the Phase II studies that the 1.10 to 1.12 Atms. TGP threshold applies not only to sub-dermal bubble growth in external epithelium tissue, but also to the growth of extracorporeal bubbles between gill lamella. However, it was not possible to demonstrate that sub-dermal bubble growth in the mouth, extracorporeal bubble growth in the gill lamella or the two acting in concert was the primary or contributing cause of mortality at the lower TGP levels. However, it was clear that in this range of TGP, arterial p 0 2 and pH declined with increasing exposure time; thus, indicating a progressive blockage of the respiratory pathway. The only apparent mechanical means for this blockage were the 180 sub-dermal bubbles in the mouth and extracorporeal bubbles in the gills. Thus there is at least a correlation of mortality, blockage of respiratory pathways and the two forms of bubble growth in this narrow range of water TGP. It was not possible to define the lower bubble growth threshold more precisely than the TGP range of 1.10 to 1.12 Atms. This was clearly due to the variability in the response of the animals. The source of this variability is not known. However, because the time to mortality is quite long in this range of TGP, factors such as fish health, handling stress and water quality are probably important considerations. Using this range (1.10 to 1.12 Atms.) for the extracorporeal and sub-dermal bubble growth thresholds, the radius of critical nuclei back calculated from Equation 6 is 12 -14)uM. 5.7.2 INTRAVASCULAR BUBBLE GROWTH THRESHOLDS: The results of the Phase II experiments clearly show the existence of a threshold for intravascular bubble growth in fish exposed to supersaturated water. This conclusion is based primarily on the observation of bubbles in the gill vasculature and the significant changes in hematocrit. A clear definition of this threshold could not be obtained from the results of the Phase II experiments. Again, the large variability in the response of the animals coupled with unknown variations in stress produced by sub-dermal and extracorporeal bubble growth clouded the response. Furthermore, as shown in the theoretical section, fish exhibit a large variation in the ratio of arterial p 0 2 to water p 0 2 (the gill uptake ratio in Equation 4). This is no doubt a contributing factor in the variability observed. Nevertheless, the results of the Series 1 through 5 experiments show that the intravascular bubble growth threshold is in the TGP range of 1.15 to 1.19 Atms. for water p 0 2 between 185 and 201 mmHg. If only the un-cannulated 181 fish of these series are considered, the threshold appears to be in a TGP range of 1.16to 1.19 Atms. The experimentally derived dependence of intravascular bubble growth thresholds on water p 0 2 can be obtained by combining the thresholds indicated in the various series (Series 1 through 5 and Series 7) with the data of Rucker (1975 and Section 5.6.4 above). The combined data are plotted in Figure 75 as a function of water p 0 2 . Shown in this plot are only data for un-cannulated fish of the Phase II studies. The arrows indicate the range of TGP and p 0 2 in which the various thresholds are indicated to lie. The individual data records are identified in the legend on the figure. Also plotted on the graph are the theoretical predictions of Equation 4 for various sizes of nucleation sites. The theoretical curves are for an oxygen uptake ratio of 0.79, a water temperature of 5 to 12 ° C. and sea level atmospheric pressure. In Figure 75, the experimental thresholds correlate well with theoretical thresholds based on a critical nuclei radius of 12 to 14 JJM. This observation is important in terms of the size of arteries in which the bubbles were found. This radius is approximately twice that of Rainbow trout red blood cells (Heming, 1984a; Mott, 1957 and Smith, 1952). As pointed out in the theoretical section, arteries of the secondary lamella have characteristic widths comparable to the diameter of red blood cells. This implies that nuclei and bubbles could not exist in the secondary lamella. As shown in the results section, with one exception, all lamella examined had bubbles only in the filamentary arteries (Figures 63 and 64). In the one gill segment that had a bubble in a secondary lamella, it was clear that its origin was in the much larger filamentary artery supplying the secondary lamella. This was observed in a single fish exposed to the highest level of TGP in the Phase II experiments. Thus, the size of nuclei is consistent with the size of gill vessels in which bubbles were found. TGP THRESHOLDS FOR BUBBLE GROWTH IN ARTERIAL BLOOD AS A FUNCTION OF WATER p02 AND NUCLEUS RADIUS 182 TGP THRESHOLD - Atms. Nucleus Radius 6 Meters * 10 1.07 50 100 150 200 250 300 350 WATER P02 - mmHg. Water Depth - 0.0 M. F - 0.79 Water Temp - 5 - 1 5 deg. C. Atmospheric Pressure • 760 mmHg. [A & B - Rainbow Trout) Fidler (1988) [C - Coho Salmon) Rucker (1975) FIGURE 75: TGP Thresholds for Bubble Growth in Arterial Blood. 183 It should be recognized that bubbles are probably growing in other locations within the animal. As mentioned in the results section above, bubbles were found in muscle, heart and liver tissue samples. Smith (1988) and Nebeker et al. (1976b) and Dawley and Ebel (1975) have also shown intravascular bubbles to be present in these organs. As with the gill vascular bubbles, the indicated size of the critical nuclei are larger than the characteristic dimensions of capillary beds. Thus, these nuclei would have to exist in vessels the size of arterioles or larger. Considering the variation of blood pressure and p 0 2 in the circulatory system (Section 3, above), the most favorable location still falls to the arterioles just upstream of the capillary beds. 5.7.3 BUBBLE GROWTH AT LOW TGP LEVELS: In the Phase II experiments, there were unique physiological characteristics associated with each form of bubble growth. In many cases these characteristics correlate with time to mortality for each bubble growth threshold. Sub-dermal Bubble Growth: This form of bubble growth took considerable time to develop at the lower levels of water TGP (1.12 to 1.15 Atms.). However, based on the size of bubbles, it appears that blisters in the buccal cavity grow more quickly than blisters formed on the opercula flaps or other external surfaces. All sub-dermal bubbles were present at TGP levels well below the thresholds for intravascular bubble growth. This implies that the gas transport pathway for this form of bubble growth is directly from the water to skin, and does not involve the circulatory system. If the circulatory system were involved, one would expect sub-dermal bubbles only at the same levels of TGP that initiate intravascular bubble growth. Furthermore, because the bubbles appear just beneath the epithelium cell layer, it is presumed that utilization of oxygen by these cells will have little effect on p 0 2 concentrations in the immediate 184 vicinity of bubble nuclei. In effect, the nuclei involved in these bubbles will see dissolved oxygen tensions close to those of the environmental water. Thus, the value of the uptake ratio (F) will be very close to 1.0. Based on the thresholds indicated for this form of bubble growth, nuclei are again about 12-14 fjM. in radius, as calculated from Equation 6. Extracorporeal Bubble Growth Between Gill Lamella: As suggested by the supplemental experiments described in the results section, these bubbles appear to develop early during exposure to supersaturated water. Furthermore, the size of these bubbles appears to be related to water TGP levels with size increasing as TGP increases. This is a reasonable conclusion since, as shown in Section 3, higher TGP levels lead to higher rates of bubble growth. Although extracorporeal bubbles were larger at higher levels of TGP, there were fewer present or they were entirely absent once TGP levels rose above 1.15 Atms. Presumably, this was because the larger bubbles could be easily dislodged by the respiratory water flow. This observation offers an explanation for the two thresholds noted in the database analysis for Chinook salmon over 50 mm. in length. It will be recalled there was a transition from one mechanism of mortality to another at a water TGP of 1.15 Atms. Furthermore, there was a sharp discontinuity in the time to mortality at this TGP. Thus, if extracorporeal bubbles are no longer present above a TGP of 1.15 Atms., sub-dermal bubbles by themselves may not be enough to cause mortality. On the other hand, if a TGP of 1.15 is the threshold for intravascular bubble growth, this mechanism of mortality may become effective above 1.15 Atms. and involve different times to mortality. Thus, a transition from one mechanism of mortality to another may in fact be occurring. 185 Epithelial Tissue Cells: It is not clear whether extracorporeal bubbles in the gills originate on nuclei associated with the epithelium tissue of the lamella or on nuclei free in the water and carried to those locations by respiration. However, the critical radius back calculated from Equation 6 is again 12 - 14 J U M . It is of interest to consider this dimension in relation to the size of tissue cells that make up the mucosal epithelial surfaces of fish gills. Laurent (1984) describes several types of cells that populate the external surfaces of Rainbow trout gills. It is significant that of these cells, both chloride and squamous cells, have characteristic dimensions on the order of 10 -12 fjM. in radius. Chloride cells appear as natural depressions in the epithelial surface. As pointed out by Harvey (1951), nuclei are frequently created by microscopic discontinuities in surfaces. Thus, based on their size and geometry, the chloride cells appear to be likely candidates for bubble nuclei. When squamous and other epithelial cells die, they are removed from the epithelial surface. The dead cells are eventually replaced by new cells that grow upward from beneath the old cell. However, before the dead cell is completely replaced, there is a temporary discontinuity in the surface about the size of the dead cell. This discontinuity may also become a temporary nucleation site for bubble growth. In Figure 28b of Laurent (1984), both chloride cells and depressions in squamous cells are clearly seen in photographs of the gill external epithelial surface. Although somewhat circumstantial, the cells that make up the external epithelial surface of the gill may, at one time or another, be nucleation sites for extracorporeal bubble growth in the gills. 186 For intravascular bubble growth, it is not known if nucleation sites are directly associated with the cells lining the circulatory system. The difficulty in establishing such a relationship is that the dimensions of these cells in fish are unknown. The only conclusion to be drawn regarding intravascular bubbles is that the dimensions of the effective nucleation site radius back calculated from the experimental data are of the same order as those of the gill epithelial cells. It is reasonable to assume that nucleation sites free in the environmental water may be carried into gill lamella by respiratory water flow. For example, water below dam spillways is often filled with bubbles. Many water sources carry a high loading of large silt particles. Thus, nucleation sites of considerable size could be carried into gill lamella and initiate extracorporeal bubble growth at very low levels of TGP. In fact, Bouck (1980) reports observing bubbles in the gills of fish exposed to TGP levels of 1.03 Atms. Unfortunately, he did not specify whether the bubbles were of intravascular or extracorporeal origin. Time to Mortality: Theory predicts that bubbles grow rapidly in the environmental water (Section 3). The Phase II experiments confirm the presence of these bubbles early in the exposure process. It is difficult, however, to explain the long time to mortality seen in the low ranges of TGP (1.10 to 1.15 Atms.) in terms of extracorporeal bubble growth alone. It appears that the time to mortality is also dependent on the much slower growing sub-dermal bubbles formed in the lining of the mouth. As mentioned in the results section, these bubbles are of a size and number that could easily block the flow of respiratory water. Although this hypothesis cannot be verified with the Phase II results, it is, at present, the most likely explanation for the times to mortality observed in the experiments. This does not imply that 187 extracorporeal bubbles are uninvolved in mortality. However, it does suggest that extracorporeal bubbles, alone, cannot produce mortality. 5.7.4 BUBBLE GROWTH AT HIGH TGP LEVELS: Intravascular Bubble Growth: The Phase II experiments demonstrate that, at high levels of TGP, bubbles form in the gill vasculature before the decline in blood pressure that precedes death. Thus, an explanation is needed for this bubble growth in the face of the high vascular system pressures in the gills. As pointed out in the discussion of Phase I results, resting fish perfuse only about 2/3 of the available lamella with blood (Farrell et al., 1979). The remaining lamella are recruited when conditions of hypoxia or exercise demanded additional oxygen. Until that time the lamella are closed off and do not respond until elevated system pressure forces them open (Farrell et al., 1979). Farrell and co-workers observed that the dormant lamella are the secondary lamella lying at the distal ends of the primary lamella. In the Phase II experiments it was noted that intravascular bubble formation also began in the distal ends of the primary lamella. Thus, if these regions are closed off, system pressures may be significantly lower than in the perfused lamella. This would allow bubble growth to begin on nuclei smaller than those required for bubble growth in the higher pressure regions of the gill vasculature (see Figure 1, Section 3). It will be recalled from Section 3 that, once a bubble begins to grow, it effectively becomes a progressively larger nucleation site. It can then continue to grow in the face of lower TGP or increased system pressure. Thus, once the critical size limitation is overcome, bubbles originating in the closed off regions of the lamella can proceed to grow into the perfused, higher pressure regions of the gill vasculature without 1 8 8 collapse. Although much of this argument is based on circumstantial evidence, it offers the most plausible explanation for the existence of these bubbles. 5.7.5 TIME TO MORTALITY: As with mortalities involving combinations of extracorporeal gill and sub-dermal buccal cavity bubble growth, it is difficult to explain the time to mortality based on the time required for intravascular bubbles to grow. As shown in Section 3, times for bubble growth are predicted to be on the order of several hours. On the other hand, except at very high levels of TGP, time to mortality is on the order of a hundred hours or more (Figure 46). There are several possible explanations for the difference between the two. Since the following explanations are not mutually exclusive, the actual cause may involve a combination of these explanations. First, it will be recalled from Section 3 that as bubble volume is added to the vascular system, there will eventually be an increase in system pressure. This was clearly evident in the Phase I experimental results. When system pressure rises, bubble growth will slow or cease completely. The continuation of growth beyond this point will depend on how rapidly water is removed from the cardiovascular system. As shown by the Phase I and Phase II hematocrit measurements, there is a net removal of water from the vascular system at the higher levels of TGP. Thus, bubble growth may be delayed by the process of water removal. As pointed out earlier, the initially rapid growth of bubbles will occur only until the bubbles block the arteries in which they are located. At that point blood flow will stop and the diffusion of gases to bubbles will be altered. For a bubble in the filamentary arteries of the primary lamella, growth is by direct diffusion of dissolved gases from the water to the bubble. Therefore, these bubbles should continue to grow even after they block the artery. As pointed out earlier, growth may continue even as the 189 bubbles advance into regions of higher system pressure. In the case of bubbles growing in tissue arteries and arterioles, growth will slow once the artery or arteriole becomes blocked. As pointed out in Section 3, once blood flow stops, the movement of dissolved gas to the bubble will be by diffusion alone through a very long diffusion pathway. Because diffusion gradients continue to decline in this process, rates of bubble growth should slow dramatically. 5.7.6 RESPONSE TO HYPOXIA: As with the Phase I studies, mortality at all levels of TGP above 1.1 Atms. seems to be related to acute hypoxia caused by blockage of respiratory pathways. The mechanisms leading to this blockage appear to be various forms of extracorporeal, sub-dermal and intravascular bubble growth. Hypoxia between TGP's of 1.10 and 1.15 Atms.: In this range of dissolved gas tension, the decline in p 0 2 appears to be the result of reduced delivery of oxygen to the blood caused by blockage of respiratory water flow. The blockage of this flow will also reduce the rate at which fish can remove C 0 2 and other metabolic wastes by way of the gills. Therefore, as blood C 0 2 concentrations increase, blood pH will decline. It has been shown that, under hypercapnia, Rainbow trout are able to recover from a similar type of respiratory acidosis. That is, with time, they are able to restore blood pH to near normal conditions (Heisler, 1984). This is accomplished through an accumulation of bicarbonate and other ionic adjustments taking place through exchange mechanisms on the gill membrane (Heisler, 1984). In the Phase II experiments, there was no evidence that fish were able to achieve this compensation. This failure to compensate may also be caused by the blockage of respiratory water flow. That is, the blockage impedes the rate of ionic exchanges that normally take place through the gill membrane. In this situation, there would be an internal buildup 190 of all metabolites including ammonia. Recently, it has been shown that ammonia facilitates the removal of CO2 (Wright et al., 1987). Ammonia excreted into the water flowing over the gill lamella provides a sink for protons produced by the ionization of carbonic acid. This causes the catalyzed CO2 - H2O reaction to favor the production of bicarbonate in the gill water boundary layer. Thus, high CO2 gradients are maintained between blood and water. A reduction in this gradient would reduce CO2 excretion by the animal and thereby lower blood pH. These conditions should exist regardless of whether reductions in oxygen uptake and metabolite excretion is the result of reduced water flow over the gills or the blockage of blood flow in portions of the gills. Response above a TGP of 1.15 Atms.: The Phase II experiments leave little doubt that bubble formation in the gill filamentary arteries occurs above a water TGP of 1.15 to 1.16 Atms. They also confirm that declines in arterial pC<2 and pH continued at the higher levels of TGP with the rate of decline increasing with water TGP. It is also clear that these declines correlate with the appearance of intravascular bubbles. At TGP levels near 1.15 Atms., extracorporeal bubbles are still present in gill lamella and may account for a portion of the p02 and pH response. However, as noted in the results, sub-dermal blisters were few or non-existent at the higher levels of TGP. Also, as in the Phase I experiments, extracorporeal bubbles became larger but less numerous as water TGP increased. As discussed earlier, the reason for fewer bubbles may be that the larger bubbles are more easily dislodged by the respiratory water flow. At TGP levels above 1.19 Atms., the number of extracorporeal bubbles were so few that it is difficult to see how they would contribute significantly to the declining arterial p02-Thus, the explanation for the declining p 0 2 may be the result of reduced blood transit time through the gill lamella. As described in the discussion of Phase I experimental 191 results, a partial blockage of the gill lamella will increase blood velocities in other lamella. If cardiac output does not fall, blood velocity will increase inversely with the fraction of arteries that are open. That is, velocity will double if half of the arteries are open or quadruple if only one fourth of the arteries are open. Randall (1982) presents information on the equilibration time for the oxygenation of blood in secondary lamella of Rainbow trout. Figure 11 of the reference shows that residence time in secondary lamella must fall below one second before there is a drop in arterial p0 2 . Randall calculates that for fully perfused secondary lamella, blood residence time in the lamella is about 3 seconds. Residence time falls to two seconds for a one third blockage of the secondary lamella. The data of Randall (1982) imply a residence time of 0.7 seconds in the secondary lamella is needed to achieve a 60% decline in arterial p02- This level of p 0 2 decline was common in the Phase II experiments. Assuming an inverse relationship between blockage and residence time, this would correspond to a blockage of at least 75% of the secondary lamella. It was observed that fish in the Series 5 experiments (TGP =1.19 Atms.), often had three quarters or more of the gill vasculature blocked by bubbles. This would correspond to a level of severity of 3 on the scale used. It will be recalled that bubbles were actually found in the filamentary arteries of the primary lamella. These bubbles would effectively block all secondary lamella at and distal to the bubble. Thus, reduced blood transit time could explain the declines in p 0 2 and perhaps the declines in blood pH as well. That is, if oxygen delivery is slowed, it would be reasonable to assume that reduced transit times would also slow the removal of C 0 2 and other metabolic wastes. Respiratory Performance: In addition to the cardiovascular response observed in the various Phase II experiments, hypoxia elicits certain characteristic respiratory adjustments in Rainbow trout. Holeton and Randall (1967a,b) and Thomas and 192 Hughes (1982) have shown that, under conditions of hypoxia, Rainbow trout will increase respiratory water flow in order to maintain oxygen delivery to the blood. This increase is accomplished by increases in both respiratory frequency and ventilation volume. The largest component of the increase is that in ventilation volume (Holeton and Randall, 1967a,b). This response was clearly observed in the Series 7 experiments where both ventilation volume and frequency increased with time (Figure 65). A comparison was made of the relationship between ventilation volume and frequency of these experiments with data from Davis and Cameron (1971), and Iwama (1986). In the Davis and Cameron experiments, respiration was examined in Rainbow trout exposed to hypoxia. Iwama looked at these parameters as a function of head differences across the respiratory system. The notable difference between the Phase II experimental data and that of Davis and Cameron and Iwama is that fish in the Series 7 experiments had lower ventilation volumes in relation to ventilation frequency. It could be concluded that the lower ventilation volumes of the Series 7 experiments were due to blockage of respiratory water flow by bubbles. However, without a comparison of hematocrit and other physiological parameters among the various fish, this conclusion is somewhat tenuous. On the other hand, the presence of extracorporeal bubbles in the gills will undoubtedly increase the resistance to water flow through the gills. This will eventually require some form of compensatory adjustment by the fish in respiration frequency and/or volume. Catacholamines: Exercising fish exhibit catacholamine levels at least an order of magnitude greater than those found in fish of the Series 6 experiments (Primmett et al., 1986). Thus, in spite of the trends shown in Figures 71 and 72, the fish do not appear to be highly stressed (as indicated by the catacholamine levels). However, other factors may prevent the measured catacholamine levels from being accurate indicators of stress. For example, with the blockage of blood flow by bubbles, it is possible that catacholamines released into the blood may not appear at the dorsal aorta. Furthermore, bubbles have been observed in the spinal columns of salmon exposed to supersaturated water (Stroud, Bouck and Nebeker, 1975). These bubbles could interfere with reflex neural pathways and inhibit the release of catacholamines. With this combination of blockage mechanisms it is not surprising that catacholamine levels were not elevated to levels characteristic of stressed fish. 6.0 CONCLUDING DISCUSSION 194 The two phases of experimental study confirm the existence of thresholds associated with various forms of bubble growth in fish exposed to supersaturated water. These thresholds correlate with thresholds for mortality observed during the experiments as well as with mortality threshold data from the literature. Furthermore, with the definition of a gill oxygen uptake ratio and a critical nucleation site radius, the thresholds can be are predicted using bubble growth threshold equations derived by this author. A mortality threshold occurring at water TGP levels between 1.10 and 1.12 Atms. corresponds to thresholds for the growth of extracorporeal gill bubbles and sub-dermal bubbles in the buccal cavity. A mortality threshold occurring at water TGP levels of 1.15 to 1.18 Atms. correlates with thresholds for intravascular bubble growth occurring in the TGP range of 1.16 to 1.19 Atms. However, in spite of these correlations, the direct implication of bubbles in the death of fish remains somewhat circumstantial. Still, several experimental observations combined with components of physiological stress measured during the experiments support the hypothesis that bubbles are the principal cause of death. First, all fish exposed to supersaturated water above a TGP of 1.12 Atms. exhibited declines in arterial p 0 2 and pH. In most cases, arterial p 0 2 measurements taken just before death showed blood p 0 2 levels were near or below those corresponding to acute hypoxia for Rainbow trout. This was at times when water p 0 2 levels were in equilibrium with atmospheric oxygen or even supersaturated with oxygen. The reductions in blood oxygen tension were clearly due to impaired movement of respiratory gases and not reduced ventilation effort on the part of the fish. Experiments with van Dam respiratory chambers showed that ventilation frequency 195 and volume were in fact elevated during exposure to supersaturated water. Detailed examination of intravascular as well as extracorporeal bubbles in gill lamella and sub-dermal bubbles in the lining of the mouth indicated that these were the only forms of mechanical blockade that could account for low arterial oxygen. Thus, the only plausible conclusion is that bubbles are involved in the hypoxic response of fish exposed to supersaturated water. The combination of extracorporeal bubbles in gill lamella and sub-dermal bubbles in the lining of the mouth appear to block respiratory water flow at TGP levels between 1.1 and 1.15 Atms. Above a TGP of 1.15 Atms., intravascular bubbles block blood flow in the filamentary arteries of gill lamella. This should produce an accelerated blood flow in the remaining unblocked arteries. Higher blood velocities reduce transit time in secondary lamella to the point where full oxygenation of the b.lood does not occur and p 0 2 levels drop. It remains unclear whether hypoxia by itself is the cause of mortality in fish exposed to supersaturated water. As indicated in the discussions of the Phase I and Phase II results, a mechanical blockage of the respiratory pathways more than likely blocks or impedes the removal of C 0 2 and ammonia from the animal. In addition, other ionic exchanges at the gill may be disrupted. Therefore, the potential exists for the physiological insult caused by hypoxia to be compounded by other factors. In addition, data from the literature show that neural functions may be disrupted by bubbles forming in the spinal columns of fish. It is conceivable that these bubbles can block the normal responses to the applied stresses and reduce survival capability even further. It should be noted that no GBT related deaths occurred that were not accompanied by decreases in arterial p02 and pH. Therefore, it is concluded that 196 hypoxia is a significant contributor to the death of fish exposed to supersaturated water. The transition from a lower TGP threshold at 1.10 to 1.12 Atms. to an upper threshold between 1.16 and 1.19 Atms. appears to involve a shift in the bubble related mechanisms that lead to mortality. At the lower threshold, sub-dermal bubbles in the lining of the mouth and extracorporeal bubbles in gill lamella appear to be the only forms of blockage to the movement of respiratory gases. As water TGP increases, both the sub-dermal and extracorporeal bubbles become larger while time to mortality decreases. However, at a water TGP above 1.15 Atms., the extracorporeal bubbles become fewer and even disappear as TGP is increased further. This appears to be the result of the larger bubbles being more easily dislodged by respiratory water flow. Furthermore, as water TGP increases above 1.15 Atms., sub-dermal bubbles in the lining of the mouth are not as large at death or are absent entirely. Apparently, this is due to the rapid onset of mortality caused by intravascular bubbles that occur at higher levels of TGP. At water TGP levels somewhere between 1.16 and 1.19 Atms., intravascular bubble formation begins and time to mortality again decreases with increasing TGP. This sequence correlates with data from the literature which suggest that during this transition there is a region of TGP that offers some relief to the fish. That is, for Chinook salmon greater than 50 mm. in length, time to mortality jumps from just a few hours at a TGP of 1.15 Atms. to about 100 hours at slightly higher levels of TGP. However, from the 100 hour level, time to mortality again declines as water TGP increases further. The transition in time to mortality may be due to the reduced number of extracorporeal gill bubbles and smaller sub-dermal bubbles in the mouth. 197 Using the threshold equations to back calculate an effective nuclei radius, it is found that a radius of 12 to 14 JJM. is common to all forms of bubble growth. It was shown that this dimension is also characteristic of epithelium cells lining the mucosal surfaces of the gill. The coincidence of these dimensions is significant and may explain the origin of nucleation sites for bubble growth. Using 12 JUM . as an effective nucleation radius, the theoretical threshold equations can be written in a final form. These are shown below as Equations 16, 17 and 18. The oxygen uptake ratio, F, has been taken as 0.79 for intravascular bubble growth and 0.85 for swimbladder overinflation. These values of F should apply for water p 0 2 levels ranging from 70 to 350 mmHg. For higher or lower water p 0 2 levels, adjustments will have to be made in the F parameter based on the data of Figure 5 . The equations have been plotted for sea level atmospheric pressure and a water temperature of 5 to 15° C . as shown in Figure 76. THRESHOLD CRITERIA FOR BUBBLE GROWTH IN THE VASCULAR SYSTEM TGPcv ^ pAtm + 73.1-h + 0.21-pO2 + 83.0 Equation 16 THRESHOLD CRITERIA FOR OVERINFLATION OF THE SWIMBLADDER T G P S B ^ PAtm + 73.1-h + 0.15-pO2 Equation 17 198 THRESHOLD CRITERIA FOR BUBBLE GROWTH IN ENVIRONMENTAL WATER T G P E W > P A t m + 73.1-h + 83.0 Equation 18 In conclusion, threshold equations 16, 17 and 18 shown plotted in Figure 76 provide useful predictive tools for persons working in the fields of fisheries, aquaculture or environmental impact assessment. For example, hyperoxic environments are now being used to increase carrying capacity of hatchery aquaculture operations. Often this is done by raising dissolved oxygen levels without compensating reductions in dissolved nitrogen levels. Therefore, water can become supersaturated with these dissolved gases. Equations 16, 17 and 18 can be used to determine the limits of oxygenation before symptoms of GBT will appear in fish. In situations where hatchery water is naturally supersaturated, the threshold equations can be used to establish criteria for the design of aeration systems to reduce dissolved gas tensions. Another example of a situation in which the threshold equations would have application involves hydroelectric dams. Often the level of supersaturation below a dam is related to the volume of water that is spilled over the dam (White et al., 1986). In some cases, minor adjustments to the spillway flow can reduce supersaturation below threshold levels and avoid harm to fish populations below the dam. As a final example, unexplained fish kills often occur in the freshwater and marine environments during periods of intense photosynthetic activity. Providing dissolved gas data are available at these times, the threshold equations will show whether or not supersaturation is a factor in these mortalities. 199 GBT TOTAL GAS PRESSURE THRESHOLDS AS A FUNCTION OF WATER p02 FOR RAINBOW TROUT TGP THRESHOLD - Atms. 50 100 150 200 250 WATER P02 - mmHg. 300 350 VASCULAR SYSTEM WATER AND SKIN SWIMBLADDER FIGURE 76: Bubble Growth Thresholds as a Function of Water p 0 2 200 REFERENCES Ackles, K.N. (1973) Proc. Symp. on Blood-bubble Interaction in Decompression Sickness. Canadian DCIEM Conf. 73-CP-960. Canadian Defense Research Board, Downsville, Ont. Adair, W.D. and Hains, J.J. (1974) Supersaturation values of dissolved gases associated with the occurrence of gas bubble disease in fish in a heated effluent. In. Thermal ecology. Eds. Gibbons, J.W and Sharitz, R.R. U.S.A.E.C. Contribution 030505. Adamson, A.W. (1967). Physical Chemistry of Surfaces. Academic Press, New York. Alderdice, D.F. and Jensen (1985) An explanation for the high resistance of incubating salmonid eggs to atmospheric gas supersaturation of water. Aquaculture 49, 85-88. Alderdice, D.F. and Jensen, J.O.T. (1985) Assessment fo the influence of gas supersaturation on salmonids in the Nechako River in relation to Kemano Completion. Can. Tech. Report Fish. Aquat. Sci. No. 1386. Alikunhi, K.H.; Ramachadran, V. and Chaudhari, H. (1951) Mortality of carp fry under supersaturation of dissolved oxygen in water. Proc. National Institute of Science, India 17, 261-264. Altman, P.L. and Dittmer, D.S. (1961). Blood and Other Body Fluids. Federation of American Societies for Experimental Biology. Altman, P.L. and Dittmer, D.S. (1964). Biological Data Book. Federation of American Societies for Experimental Biology. Altman, P.L. and Dittmer, D.S. (1971). Respiration and Circulation. Federation of American Societies for Experimental Biology. Baroin, A.; Garcia-Romeu, F.; Lamarre, T. and Motais, R. (1984) A transient sodium-hydrogen exchange system induced by catacholamines in erythrocytes of rainbow trout, J. Physiol. (London), 356, 21-31. Becker, C D . (1973) Columbia river thermal effects study: reactor effluent problems. Journal Of the water pollution control Fed. 45, 850-869. Beiningen, K.T. and Ebel, W.J. (1970) Effect of the John Day Dam on dissolved nitrogen concentrations and salmon in the Columbia river. Trans. Am. Fish. Soc. 99, 664-671 Berg A. et al. (1984) Supersaturation of dissolved air in the waterways of hydroelectric power plants: causal relationships, detrimental effects and preventative measures. Norwegian Hydrodynamics Laboratories. Beyer, D.; D'Aoust, B.G. and Smith, L. (1976a) Response of Coho salmon to supersaturation at one atmosphere, in Fickeisen and Schneider (1976) 47-50 Beyer, D.; D'Aoust, B.G. and Smith, L. (1976b) Decompression induced bubble formation in Salmonids. Comparison to gas bubble disease. Undersea Biomedical Res. 3,321-338 Bird, R.B.; Stewart, W.E. and Lightfoot, E.N. (1960). Transport Phenomena. John Wiley & Sons, New York. Black, E.C. and Irving, L. (1937) The effect of carbon dioxide upon oxygen capacity of the blood of the carp. Trans. R. Soc. Can. Sect. 5, 29-32. Blahm, T.H.; McConnell, R.J. and Snyder, G.R. (1973) Effect of gas supersaturated Columbia River water on the survival of juvenile salmonids. Final Report - Part 1. Nat. Mar. Fish. Serv. Environmental Field Station. Prescott, Oregon. Blahm, T.H.; McConnell, R.J. and Snyder, G.R. (1975) Effect of gas supersaturated Columbia River water on the survival of juvenile Chinook and Coho salmon. NOAA Tech. Rept. NMFS SSRF-688. 202 Booth, J.H. (1980) The effects of oxygen supply, epinephrine, and acetylcholine on the distribution of blood flow in trout gills. J. exp. Biol. 83, 31-39 Bouck, G.R.; Chapman, G.A.; Schneider, P.W. Jr. and Stevens, D.G. (1970) Observations on gas bubble disease in adult Columbia River sockeye salmon. Pacific Northwest Laboratory, Federal Water Quality Administration, Corvalis, Oregon. Bouck, G.R. (1980) Etiology of gas bubble disease. Trans. Am. Fish. Soc. 109, 703-707. Bouck, G.R. (1982) Gasometer: an inexpensive device for continuous monitoring of dissolved gases and supersaturation. Trans. Am. Fish. Soc. 111, 505-516. Boutilier, R.G.; Heming, T.A. and Iwama, G.K. (1984). Physicochemical parameters for use in fish respiratory physiology. In Fish Physiology, Vol. Xa. Edited by W.S. Hoar and D.J. Randall, Academic Press. 403-430. Bowser, P.R.; Toal, R.; Robinette, H.R. and Brunson, M.W. (1983) Coelomic distension in channel catfish fingerlings. Prog Fish. Cult. 45, 208-209 Boyer, P.B. (1974) Lower Columbia and lower Snake Rivers, nitrogen gas supersaturation and related data analysis and interpretations. U.S. Army Corps of Engineers, North Pacific Division, Portland Oregon. Boyle, R. (1670) New pneumatical observations concerning respiration. Philosophical Transactions. 5:2011-2031, 2035-2056. Cameron, J.N. (1971) Oxygen dissociation characteristics of the blood of rainbow trout, Comp. Biochem. Physiol. 38A, 699-704. Cameron, J.N. and Davis, J.C. (1970) Gas exchange in rainbow trout (Salmo Gairdneri) with varying blood oxygen capacity. J. Fish. Res. Board. Can. 27, 1069-1085. 203 Cameron, J.N. and Randall, D.J. (1972) The effect of increased ambient C 0 2 tension, C 0 2 content and pH in rainbow trout. J. exp. Biol. 57, 673-680. Casillas, E.; Miller, S.E.; Smith, L.S. and D'Aoust, B.G. (1975). Changes in hemostatic parameters in fish following rapid decompression. Undersea Biomedical Research. 2:267-276. Casillas, E.; Smith, L.S. and D'Aoust, B.G. (1976a). The response of fish blood cells, particularly thrombocytes, to decompression. Undersea Biomedical Research. 3:273-281. Casillas, E.; Smith, L.S. and D'Aoust, B.G.( 1976b) Effects of stress on salmonid blood clotting mechanisms, in Fickeisen and Schnider (1976) 93-95 Champeney, D.C. (1973). Fourier Transforms and their physical applications. Academic Press, New York. Colt, J.E. and Cornacchia, J.W. (1984). The effects of dissolved gas supersaturation on larval striped bass, Morone saxatilis (Walbaum). J. Fish dis. 7:15-27. Colt, J.E. (1983). The computation and reporting of dissolved gas levels. Water Res. 17:841-849. Colt, J.E. (1984) Computation fo dissolved gas concentrations in water as functions fo temperature, salinity and pressure. Special publication No. 14, Amer. Fish. Soc, Bethesda Md. Colt, J.E.; Bouck, G and Fidler, L.E. (1986) Review of current literature and research on gas supersaturation and gas bubble trauma. U.S. Dept. of Energy. Bonneville Power Admin. Div. of Fish and Wildlife. Cornacchia, J.W. and Colt, J.E. (1984) The effects of dissolved gas supersaturation on larval striped bass, Morone saxatilis. J. Fish Diseases. 7, 15-27. 204 Clift, R.; Grace, J.R. and Weber, M.E. (1978). Bubbles, Drops and Particles. Academic Press, New York. Coutant, C.C. and Genoway, R.B. (1968) Final report on the exploratory study of interaction of increased temperature and nitrogen supersaturation on the mortality of adult salmonids. U.S. Bur. Commercial Fish. Seattle, PAC N.W. Lab. Richland Wash. Davis, J.C. and Cameron, J.N. (1971) Water flow and gas exchange at the gills of rainbow trout (Salmo gairdneri). J. exp. Biol. 54, 1-18. Dawley, E.M. and Ebel, W.J. (1975) Effects of various concentrations of dissolved gas on juvenile chinook salmon and steelhead trout. U.S. Fish. Bull. 73, 787-796. Dawley, E.M.; Schiewe, M. and Monk, B. (1976). Effects of long term exposure to supersaturation of dissolved atmospheric gases on juvenile Chinook salmon and Steelhead trout in deep and shallow tank tests. In: Fickeisen & Schneider (1976): 1-10. Dawley, E.M. and Ebel, W.J. (1975). Effects of various concentrations of dissolved atmospheric gas on juvenile chinook salmon and steelhead trout. United States National Marine Fisheries service Fishery Bulletin. 73:787-796. Dell, M.B.; Erho, M.W. (1975) Occurrence of gas bubble disease symptoms on fish in mid Columbia reservoirs. Grant, Douglas, and Chelan County Public Utility Districts. Ephrata and Wenatchee, Washington. DeMont, J.D. and Miller, R.W. (1972) First reported incidence of gas bubble disease in the heated effluent of a steam electric generating station. Proc. of the Annual Conf. S.E. assoc. of Game and Fosh Comm. 25, 392-399 D'Aoust, B.G. and Smith, L.S. (1974) Bends in fish. Comp. Biochem. and Physiol. 49, 311-321 Ebel, W.J. (1969) Supersaturation of nitrogen in the Columbia River and its effect on salmon and steelhead trout. U.S. Natl. Marine Fish. Serv. Bulletin 68, 1-11 205 Ebel, W.J.( 1971) Dissolved nitrogen concentrations in the Columbia and Snake Rivers in 1970 and their effect on chinook salmon and steelhead trout. NOAA. Tech. Report. NMFS SSRF 646. Ebel, W.J. (1979) Effects of atmospheric gas supersaturation on survival of fish and evaluation of proposed solutions. 5th. Prog Report, U.S. Army Corps of Engineers. Ebel, W.J.; Dawley, E.M. and Monk, B.H. (1971) Thermal tolerance of juvenile salmon in relation to nitrogen supersaturation. U.S. fish and Wildlife Service Fisheries Bulletin. 69, 833-843. Ebel, W.J.; Kroma, R.W. and Raymond, H.L. (1973) Evaluation offish protective facilities at Little Goose Dam and review of other studies relating to protection of juvenile salmonids in the Columbia and Snake Rivers, 1973. National Marine Fisheries Service. Northwest Fisheries Center, Seattle Washington. Ebel, W.J.; Raymond, H.L. Monan, G.E.; Farr, W.E. and Tanonaka, G.K. (1975) Effect of atmospheric gas supersaturation caused by dams on salmon and steelhead trout of the Snake and Columbia Rivers. National Marine Fisheries Service. Northwest Fisheries Center, Seattle Washington. Ebel, W.J.; Beningen, K.T.; Bouck, G.R.; Penrose, W.R. and Weitkamp, D.E. (1979) Gases, total dissolved. In: Thurston, R.V.; Russo, R.C.; Fetterolf, C M . , Edsall, T.A. and Barber, Y.M. editors. A review of the EPA red book: quality criteria for water. American Fisheries Soc. Ebeling, G. (1954) Sauerstoff-Ubersattigung in fliessender und stehenden Gewassern Vom Wasser. 21, 84-99. Eckert, R. (1983) Osmoregulation and excretion. In: Animal Physiology, Eckert, R. and Randall, D.J.. W.H. Freeman, San Francisco: 483-543. Eddy, F.B.; Lomholt, J.P.; Weber, R.E. and Johansen, K. (1977) Blood respiration properties of rainbow trout kept in water of high C 0 2 tension. J.exp Biol. 67, 37-47. 206 Epstein, P.S. and Plesset, M.S. (1950). On the stability of gas bubbles in liquid gas solutions. J. Chem. Phy. 18(11):1505-1509. Fairbanks, R.B. and Lawton, R.P. (1977) Occurrence of large striped mullet in Cape Cod Bay, Massachusetts. Chesapeake Sci. 18, 309-310. Fange, R. (1966) Physiology of the swimbladder. Physiol Rev. 46, 299-322. Farrell, A. P. (1988) Personal Communication. Farrell, A.P.; Sobin, S.S.; Randall, D.J. and Crosby, S. (1980) Intralamellar blood flow patterns in fish gills. Am. Jour, of Physiol. 239, 428-436. Farrell, A.P.; Daxboeck, C. and Randall, D.J. (1979) The effect of input pressure and flow on the pattern and resistance to flow in the isolated perfused gill of a teleost fish. Jour. Comp. Physiol. 133, 233-240 Feathers, M.G. and Knable, A.E. (1983) Effects of depressurization upon largemouth bass. North Amer. J. Fish. Man. 3, 86-90 Feigl, E.O. (1974) Physics of the cardiovascular system. In: Ruch, T.C. and Patton, H.D. eds. Physiology and Biophysics. 20th. ed. Vol.2 Philadelphia: Saunders. Ferguson, J.K.W. and Black, E.O (1941) The transport of C 0 2 in the blood of certain fresh water fishes. Biol. Bull. 80, 139-152. Fickeisen, D.W. and Schneider, J.M. (1976). Gas Bubble Disease. CONF - 741033. Technical Information Center, Energy Research and Development Administration, Oak Ridge, Tennessee, USA. Fidler, L.E. (1983) Design and analysis methods for hatchery aerations systems. Report to Dept. Fisheries and Oceans, Canada. Salmon Enhancement Program. Fidler, L.E. (1985). Biophysical phenomena associated with gas bubble trauma in fish. M.Sc. Thesis, University of British Columbia, Vancouver, B.C., Canada. 207 Folkow, B. and Neil, E. (1971). Circulation. Oxford University Press. Foster, R.E. and Steen, J.B. (1969) The rate of the Root shift of ell red cells and haemoglobulin solution. J. Physiol. 204, 259-282. Harvey, H.H. and Cooper, A.C. (1962) Origin and treatment of a supersaturated river water. Progress Report 9., Intnl. Pacific Salmon Fisheries Commission., Vancouver, Canada. Harvey, E.N.; Barnes, D.K.; McElroy, W.D. Whiteley, A.H; Pease, D.C. and Cooper, K.W. (1944) Bubble formation in animals. Jour. Cell, and Comp. Physiol. 24, Harvey, E.N. (1951) Physical factors in bubble formation. In: Decompression sickness. Ed. F.J. Fulton. Saunders, Philadelphia. Harvey, H.H. (1967) Supersaturation of lake water with a precaution to hatchery usage. Trans. Am. Fish Soc. 96, 194-201. Harvey, H.H. (1975). Gas disease in fish - a review. In: Chemistry and physics of aqueous solutions. Edited by W.A. Adams. The Electrochemical Society, Princeton, New Jersey. 450-485. Harvey, H.H (1963) Pressure in the early life stage of salmon. Ph. D. Thesis. The University of British Columbia. Dept. of Zoology. Hauck, K. (1986) Gas bubble disease due to helicopter transport of pink salmon. Trans. Am. Fish. Soc. 115, 630-635 Heggberget, T.G. (1984) Effect of supersaturated water on fish in river Nidelva, southern Norway. J. Fish. Biol. 24, 65-74 Heisler, N. (1984). Acid - base regulation in fishes. In: Fish Physiology, Vol X. Edited by W.S. Hoar and D.J. Randall. Academic Press. 315-401. 208 Heming, T.A.: (1984 a). The role of fish erythrocytes in transport and excretion of carbon dioxide. Ph.D. Thesis, University of British Columbia, Vancouver, B.C. Heming, T.A.: (1984 b). Unpublished Data. Dept. of Zoology, University of British Columbia, Vancouver, B.C. Heming, T.A.; Randall, D.J. and Mazeaud, M.M. (1987) Effects of adrenaline on ionic equilibria in red blood cells of rainbow trout. Fish Physiol. Biochem. 3(2):83-90. Hemmingsen, B.B.; Stienberg, N.A. and Hemmingsen, E.A. (1985) Intracellular gas supersaturation tolerances of erythrocytes and research ghosts. Biophys. J., 47, 491-496. Hemmingsen, B.B. (1986) Promotion of gas bubble formation by ingested nuclei in ciliate, Tetrahymena pyriformis. Cell Biophysics. 8, 189-200 Hills, B.A. (1977) Decompression Sickness; John Wiley & Sons. New York. Hobe, H; Wood, C M . and Whetly, M.G. (1984) The mechanisms of acid-base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. Resp. Physiol. Holl, K. (1955) Chemische Untercuchungen an kleinen Fliessgewassern. International Association of theoretical and applied limnology proceedings. 12, 360-372. Holmes, W.N. and Donaldson, E.M. (1969). The body compartments and the distribution of electrolytes. In: Fish Physiology, Vol. I. Edited by W.S. Hoar and D.J. Randall, Academic Press. 1-79. Holton, G.F. (1972). Gas exchange in fish with and without hemoglobin. Respir. Physiol. 14:142-150. Holton, G.F. (1980). Oxygen as an environmental factor of fishes. In: Environmental Physiology of Fishes. Edited by M.A.AIi. Plenum, New York. 7-32. 209 Holeton G.F. and Randall, D.J. (1967a) The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout. J. exp. Biol. 46, 317-327. Holeton G.F. and Randall, D.J. (1967b) Changes in blood pressure in the rainbow trout during hypoxia. J. exp. Biol. 46, 297-305. Hsieh, D.Y. (1965). Some analytical aspects of bubble dynamics. J. Basic Eng. Trans. ASME. Vol. 87 No. 4:991-1005. Isaia, J. (1984) Water and nonelectrolyte permeation. In: Fish Physiology Vol 10B. eds. Hoar, W.S. and Randall, D.J., Academic Press. Iwama, G. (1986) Strategies for acid-base regulation in fishes. Ph. D. Thesis. The University of British Columbia. Dept. of Zoology. Jarnefelt, V.H. (1948) Der Einfluss der Stromschnellen auf den Sauerstoff und Kohlensauregehalt und das pH des Wassers im Flusse Vuoski. International association of theoretical and applied limnology proceedings. 10, 210-215 Jensen, J. (1980). Effect of TGP and total water hardness in Steelhead eggs and alevins. A progress report. Proc. N. W. Fish Culturists Conf. Courtney, B.C., Canada. 15-22. Jensen, J.O.T.; Halley, A.N. and Schnute, J. (1985) Literature data on salmonid response to gas supersaturation and ancillary factors. Can. Data Report of Fish, and Aquatic Sci. No. 501 Jensen, J.O.T.; Schnute, J. and Alderdice, D.F. (1986). Assessing juvenile salmonid response to gas supersaturation using a general multivariant dose - response model. Can. J. Aqua. Sci. 43:1694-1709. Johansen. K. and Hansen.D. (1967) Functional anatomy of the hearts of lungfish and amphibians. Am. Zool. 8, 191-210 Johnson, D.W and Katavic, I. (1984) Mortality, growth and swimbladder stress syndrome of sea bass larvae under varied environmental conditions. Aquaculture. 38 67-78. Jones, D.R. and Randall, D. J. (1978). The respiratory and circulatory systems during exercise. In: Fish Physiology, Vol. VII. Edited by W.S. Hoar and D.J. Randall, Academic Press. 425-501. Kiceniuk, J.W. and Jones, D.J. (1977). The oxygen transport system in trout during sustained exercise. J. exp. Biol. 69:247-260. Kiceniuk, J.W. (1969) Unpublished Data. Kirsch, R. and Nonnote, G. (1977) Cutaneous respiration in three freshwater teleosts. Resp. Physiol. 29, 339-354 Knittel, M.D.; Chapman, G.A. and Garton, R.R. (1980) Effects of hydrostatic pressure on steelhead survival in air supersaturated water.Trans. Am. Fish. Soc. 109, 755-759 Kolbeinshavn, A. and Wallace, J.C. (1985) Observations of swimbladder stress syndrome in arctic char induced by inadequate water depth. Aquaculture. 36, 259-261. Kraul, S. (1983) Results and hypothesis for the propagation of the grey mullet. L. Aquaculture. 30, 273-284. Laurent, P. (1984) Gill internal morphology. In: Fish Physiology- Vol 10A. Eds. Hoar, W.S. and Randall, D.J., Academic Press. Lindroth, A. (1957) Abiogenic gas supersaturation of river water. Archiv. fur Hydrobiologie. 53, 589-597. Makren, A. (1974). Hemoglobins, myoglobins and haptoglobins. In: Clinical Chemistry - Principles and Technics. 2nd. ed. Edited by: R.F. Henry, D.C. Cannon and J.W. Winkelman. Harper and Row. 1128-1135. 211 Marcello, R.A. Jr. and Fairbanks, R.B. (1976) Gas bubble disease in Atlantic menhaden at a coastal nuclear power plant. In: Fickeisen and Schneider (1976). Marsh, M.C. (1910) Notes on the dissolved gas content of water in its effects upon fish. Bull. U.S. Bureau of Fisheries. 28, 891-906 Matsue, Y.; Egusa, S.and Saeki (1953) On nitrogen gas contents dissolved in flowing water of artesian wells and springs. Bull. Japanese Soc. Sci. Fish. 19, 439-444. Mazeaud and Mazeaud (1981) Adrenergic responses to stress in fish. In: Stress in Fish, Ed. A.D, Pickering. Academic Press. Meekin, T.A. and Turner, B.K. (1974). Tolerance of Salmonid eggs, juveniles and squawfish to supersaturated nitrogen. Wash. Dept. of Fisheries Tech Rept. 12:78-126. Meisel, S.; Nir, A. and Kerem, D. (1981) Bubble dynamics in perfused tissue undergoing decompression. Resp. Physiol. 43, 89-98. Miller, R.W. (1974) Incidence and cause of gas bubble disease. In: Gibbons, J.W. and Sharitz, R.R., Ed. Thermal Ecology. Conf. 730505 U.S. EPA, Washington, D.C. Mott, J.C.: (1957). In: The Physiology of Fishes; Edited by M.E. Brown. Academic Press, New York. Nakano and Tomlinson (1968) Catacholamine concentrations in rainbow trout, Salmo gairdneri in relation to physical disturbances. J. Fish. Res. Bd. Can. 25, 603 Nebeker, A. V.; Bouck, G.R. and Stevens, D.G. (1976) Carbon dioxide and oxygen-nitrogen ratios as factors affecting salmon survival in air supersaturated water. Trans. Am. Fish. Soc. 105, 425-429. Nebeker, A.V.; Stevens, D.G. and Baker, R.J (1976b) Survival of salmon smolts in sea water after exposure to air supersaturated water. Prog. Fish-Cult. 41, 30-31. Nebeker. A.V.; Hauck, A.K. and Baker, R.J. (1979) Temperature and oxygen-nitrogen gas ratios affecting fish survival in air supersaturated water. Water Res. 13, 299-303. Nebeker, A.V.; Stevens, D.G. and Stroud, R.K. (1976) Effects of air supersaturated water on adult sockeye salmon. J. Fish. Res. Board. Ca. 33, 2629-2633. Nebeker, A.V.; Andros, J.D.; McCrady, J.K. and Stevens, D.G. (1978) Survival of steelhead trout eggs, embryos, and fry in air supersaturated water. J. Fish. Res. Board Can. 35, 261-264. Nebeker, A.V. and Brett. J.R. (1976) Effects of air supersaturated water on survival of Pacific salmon and steelhead smolts. Trans. Am. Fish. Soc. 105, 338-342. Newcomb, T.W. (1976). Changes in blood chemistry of juvenile steelhead, Salmo gairdneri, following sublethal exposure to nitrogen supersaturation. In: Fickeisen & Schneider (1976): 96-100. Nikinmaa, M. (1982) The effects of adrenaline on red cell volume and concentration gradient of protons across the red cell membrane in the rainbow trout. Mol. Physiol., 71 A, 353-356. Nikinmaa, M. and Huestis (1884) Adrenergic swelling in nucleated erythrocytes: Cellular mechanisms in a bird, domestic goose and two teleosts, striped bass and rainbow trout. J. exp. Biol., 113, 215-224. Perry, R.H. and Chilton, C.H. (1973). Chemical Engineers Handbook. 5th. ed. McGraw Hill, New York. Peterson, H. (1971). Smolt rearing methods, equipment and techniques used successfully in Sweden. Atlantic Salmon Foundation Special Publication Series. 2(1):32-62. Philp, B.; Inwood, M.J., and Warren, B.A. (1972). Interactions between gas bubbles and components of the blood: Implications in decompression sickness. Aerospace Medicine, Sept. 1972. 946-953. Piper, J. and Scheid, P. (1984) Model analysis of gas transfer in fish gills. In: Fish Physiology Volume 10A. eds. Hoar, W.S. and Randall, D.J., Academic Press. Plesset, M.S. and Zwick, S.A. (1954). The growth of vapor bubbles in superheated liquids. J. Appl. Physics. Vol. 25, No. 4. Plesset, M.S. (1964). In: Cavitation in real fluids. Edited by R. Davis. American Elsevier Co., New York. Primmett, D.N.R.; Randall, D.J.; Mazeaud, M.M. and Boutilier, R.G. (1986) The role of catacholamines and oxygen transport in rainbow trout {Salmo gairdneri). J. exp. Biol. 123, 139-148. Rabiner, L.R. and Gold, B. (1975). Theory and application of digital signal processing. Prentice Hall, Englewood Cliffs, N.J. Railo, E.; Nikinmaa, M. and Soivio, A. (1985). Effects of sampling on blood parameters in Rainbow trout, Salmo gairdneri. J. exp. Biol. 126:489-512. Randall, D.J. (1982). Blood flow through gills. In: Soc. Exp. Biol. Sem. Series 16, Gills. (Ed. D.F. Houlihan; Rankin, C. and Shuttleworth, T.J.). Cambridge University Press. 173-191. Randall, D.J. (1984). Oxygen and carbon dioxide transfer across fish gills. In: Fish Physiology, Vol X. Edited by W.S. Hoar and D.J. Randall. Academic Press. 263-314. Randall, D.J. (1970 a). Gas exchange in fish. In: Fish Physiology, Vol IV. Edited by W.S.Hoar and D.J. Randall. Academic Press. 253-292. Randall, D.J. (1970 b). The circulatory system. In: Fish Physiology, Vol IV. Edited by W.S. Hoar and D.J. Randall. Academic Press. 133-172. Randall, D.J. and Jones, D.R. (1973). The effect of deafferentation of the pseudobranch on the respiratory response to hypoxia and hyperoxia in the trout. Respiratory Physiology. 17:291-301. Randall, D.J. and Shelton, G. (1963). The effects of changes in environmental gas concentrations on the breathing and heart rate of teleost fish. Comp. Biochem. Physiol. 9:229-239. Randall, D.J. and Daxboeck, C. (1984) Oxygen and carbon dioxide transfer across fish gills. In: Fish Physiology, Vol. XA. Eds. Hoar, W.S. and Randall, D.J. Academic Press. 263-314. Reid, R.C,; Prausnitz, J.M. and Sherwood, T.K. (1977). The properties of Gasesand Liquids. 3rd. ed. McGraw Hill Co., New York. Reintjes, J.W. (1969) Synopsis of biological data on the Atlantic menhaden. Circular 320, U.S. Fish and Wildlife Service. Renfro, W.C. (1963). Gas bubble mortality of fishes in Galveston Bay, Texas. Trans. Amer. Fish. Soc. 92:320-322. Riggs, A. (1979) Studies of the hemoglobins of Amazonian fishes, an overview. Comp. Biochem. Physiol. 62A, 257-271. Rosenthal, H. (1988) personal communication. Rucker, R.R. (1975a) Gas bubble disease mortalities of coho salmon in water with constant total gas pressure and different oxygen-nitrogen ratios. U.S. fish. Bull. 73, 915-918. Rucker, R.R. (1975b) Excess nitrogen gas in water not a cause of coagulated yolk disease in chinook salmon. Prog. Fish cult. 37, 101-102. Rucker, R.R and Turtle, E.M. (1948) Removal of excess nitrogen in a hatchery water supply. Prog. Fish Cult. 10, 88-90. 215 Rucker, R.R. and Kangas, P.M. (1974) Effect of nitrogen supersaturated water on coho and Chinook salmon. Prog. Fish Cult. 36, 152-159. Rukavina, J. and Varenika, D. (1956) Air bubble disease of trout of the source of the river Bosna Acta Ichthyologica Bosniae et Hercegovinae 1(7) 5-12. Schiewe, M.H. (1974). Influence of dissolved atmospheric gas on swimming performance of juvenile Chinook salmon. Trans. Amer. Fish. Soc. 103:717-721. Schmassermann, H. (1951) Untersuchungen uber den Stoffhaushalt Gewasser. Schweizerische Zeitschrift fur Hydrologie. 13, 300-335. Schnute, J. and McKinnell (1984). A biological meaningful approach to response surface analysis. Can. J. Fish. Aquat. Sci. 41:6. Shelton, G., Jones, D.R. and Milsom, W.K. (1986) Control of breathing in ectothermic vertebrates. In: Handbook of Physiology - The respiratory system II, American Physiological Society. 857-909. Shirahata, S. (1966). Experiments on nitrogen gas disease with Rainbow trout fry. Bulletin of the Freshwater Research Laboratory, Tokyo. 15:197-211. Shrimpton, M.J.; Randall, D.J. and Fidler, L.E. (1988). Factors affecting swimbladder volume in Rainbow trout (Salmo gairdneri) held in gas supersaturated water. (In preparation). Smith, C.E. (1988) Histopathology of gas bubble disease in juvenile Rainbow trout. U.S. Fish and Wildlife Service. Fish Technology Center, Bozeman, Montana, in press. Smith, F.M. and Jones, D.R. (1982) The effect of changes in blood oxygen carrying capacity on ventilation volume in the Rainbow trout {Salmo gairdneri). J.exp. Biol. 97, 325-334. Smith, C.G.; Lewis, W.M. and Kaplan, H.M. (1952). Comparative morphologic and physiologic study of fish blood. Prog. Fish Cult. 14:169-172. 216 Soivio, A.; Westman, K. and Nyholm, K. (1972). Improved methods of dorsal aorta catheterization: Haematological effects followed for three weeks in Rainbow trout, Salmo gairdneri. Finnish Fish Res. 1:11-12. Sovio, A; Nikinmaa, M.; Nyholm, K. and Westman, K. (1981) The role of gills in the responses of (Salmo gairdneri) during moderate hypoxia. Comp. Biochem. Physiol. 70A, 133-139. Sovio, A.; Westman, K. and Nyholm, K. (1972) Improved methods of dorsal aorta catheterization, Haemotological effects followed fro three weeks in Rainbow trout (Salmo gairdneri). Finnish Fish. Res. 1, 11-21. Sprague, J.B. (1969) Measurement of pollutant toxicity to fish. I. bioassay methods for acute toxicity. Water Res. 3, 793-821. Spry, D.J. and Wood, C M . (1985) Ion flux rates, acid-base status, and blood gases in Rainbow trout (Salmo gairdneri) exposed to toxic zinc in natural soft water. Can. J. Fish. Aquat. Sci. 42, 1332-1341. Steen, J.B. (1970) The swim bladder as a hydrostatic organ. In: Fish Physiology Vol. 4., eds. Hoar, W.S. and Randall, D.J., Academic Press. Steen, J.B. and Sund, T. (1977). Gas deposition by counter-current multiplication in the ell swim-bladder: Experimental verification of a mathematical model. J. Physiol. 267: 697-702. Stevens, E.D. (1968) Cardiovascular dynamics during swimming in fish, particularly Rainbow trout (Salmo gairdneri) Ph. D. thesis, The University of British Columbia. Dept. of Zoology. Stevens, E.D. and Randall, D.J. (1967). Changes of gas concentrations in blood and water during moderate swimming activity in Rainbow trout. J. exp. Biol. 46:307-315. Stroud, R.K. and Nebeker, A.V. (1976) A study of the pathogenesis of gas bubble disease in steelhead trout. In: Fickeisen and Schneider (1976) Stroud, R.K.; Bouck, G.R. and Nebeker, A.V. (1975) Pathology of acute and chronic exposure of salmonid fishes to supersaturated water. In: Adams, W.A., Editor. Chemistry an physics of aqueous gas solutions. The Electrochemical Society. Princeton, N.J., U.S.A. Supplee, V.G. and Lightner, D.V. (1976) Gas bubble disease due to oxygen supersaturation in raceway reared California brown shrimp. Prog. Fish Cult. 28, 198-199. Thomas, S. and Hughes, G.M. (1982) A study of the effects of hypoxia on acid-base status of rainbow trout blood using an extracorporeal blood circulation. Resp. Physiol. 49, 371-382. Tuurala, H. (1983) Structure and blood circulation of the secondary lamella of [Salmo gairdneri) gills in relation to oxygen transfer. University of Helsinki, Dept. of Zool/Physiol. Tufts, B. L. (1988) Ion exchange mechanisms for the control of volume and pH in fish and amphibian erythrocytes. Ph. D. Thesis. The University of British Columbia. Dept. of Zoology. Van, R.D. and Clark, H.G. (1975) Bubble growth and mechanical properties of tissue in decompression. Undersea Biomedical Research. 2(3), 185-194. Warren, C. and Doudoroff, P. (1971) Biology and water pollution control. W.B. Saunders Co. Toronto, Ont., 434. Weber, R.F. (1982) Intraspecific adaptations of hemoglobin function in fish to oxygen availability. In: Exogenous and Endogenous Influences on metabolic and neural Control, eds. Addink, A.D.F. and Sprank, N. 87-102, Pergamon Press. Weiss, R.F. (1970). The solubility of nitrogen, oxygen and argon in water and sea water. Deep Sea Research. 17:721-735. 218 White, R.G., Phillips, G. and Liknes, G. (1986). The effects of supersaturation of dissolved gases on the fishery of the Bighorn river downstream of the Yellowtail afterbay dam. Montana Cooperative Fishery Research Unit, Montana State University, Bozeman Montana. Weitkamp, D.E. and Katz, M. (1980). A review of dissolved gas supersaturation literature. Trans. Am. Fish. Soc. 109:659-702. Weitkamp, D.E. (1974) Dissolved gas supersaturation in the Columbia River system: salmonid bioassay and depth distribution studies, 1973 and 1974. Parametrix, Inc. Report to Utility Cooperative, Idaho Power Company., Boise, Idaho. Weitkamp, D.E. (1976) Dissolved gas supersaturation: live cage bioassays of Rock Island Dam, Washington. In Fickeisen and Schneider (1976) Westman, J.R. and Nigrelli, R.F. (1955) Preliminary studies fo menhaden and their mass mortality in Long Island and New Jersey waters. New York Fish and Game J., 2: 142-153. White, R.G.; Phillips, G.; Liknes, G. and Sanford, S. (1986) The effects of supersaturation of dissolved gases on the fishery of the Bighorn River downstream of the Yellowtail Dam. Annual report to U.S. Bureau of Reclamation. Wolf, K. (1963) Physiological salines for freshwater teleosts. Prog Fish Cult. 25, 135-140. Wood, C M . and Jackson, E.B. (1980). Blood acid base regulation during environmental hypoxia in the Rainbow trout, Salmo gairdneri. Respir. Physiol. 42:351-372. Wood, C M . ; Wheatly, M.G. and Hobe, H. (1984) The mechanisms of acid base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. III. Branchial exchanges. Respir. Physiol. 55, 175-192. 2 1 9 Woodbury, L.A. (1941). A sudden mortality of fishes accompanying a supersaturation of oxygen in Lake Waubesa, Wisconsin. Trans. Amer. Fish. Soc. 71:112-117. Wright, P.A., Heming, T.A. and Randall, D.J. (1986). Downstream pH changes in water flowing over the gills of Rainbow trout. J. exp. Biol. 126:489-512. Wright, P.A. and Randall, D.J. (1987) Ammonia distribution in fish. Fish Physiol. Biochem. 3 (3), 107-120. Wyatt, E.J. and Beiningen, K.T. (1969) A nitrogen gas disease catastrophe. Proceedings the 20th. annual northwest fish culture conference. 71-72. Yamamoto, K.I.; Itzawa, Y. and Kobayashi, H. (1980) Supply of erythrocytes into the circulatory blood from the spleen of exercising fish. Comp. Biochem. Physiol. 65A, 5-11. Yang, W.J. and Liang, C.Y. (1972) Dynamics of the dissolution of gas bubbles or pockets in tissue. J. Biochem. 5. Yount, D.E. (1979) Application of a bubble formation model to decompression sickness in rats and humans. Aviation, Space and Environmental Medicine. January. Yount, D.E. and Yeung, C M . (1979) Determination of the radii of gas cavitation nuclei by filtering gelatin. J. Acoustic Soc. Am. 65 (6). Yount, D.E. (1981) A model for microbubble fission surfactant solutions. J. Colloid and Interfacial Science 91 (2) Zaitsev, Y.P. (1971) Marine Neustonology, Israel Program for Scientific Translations, Jerusalem. APPENDIX A DERIVATION OF BUBBLE GROWTH THRESHOLD EQUATIONS 220 This derivation begins with the development of an equation for the growth of bubbles in the cardiovascular systems of fish exposed to supersaturated water. In the derivation, it will be assumed that bubble growth occurs under isothermal conditions and that inertial effects are negligible (i.e. the growth process is diffusion limited -Hseih, 1965). Furthermore, it will be assumed that the diffusing gases are oxygen, nitrogen and water vapor and that they obey the perfect gas law. In addition, it will be assumed that the principal resistance to gaseous diffusion lies in the water or plasma phase, that the interior of the bubble is of uniform composition and the gas and liquid phases are inviscid. Finally, the partial pressure of water vapor in the bubble will be taken as the saturated vapor pressure of water at the isothermal temperature. By the perfect gas law the number of moles of oxygen in a bubble (ngb) within the vascular system is given by: 4 - 7 r r 3 p o b nOb = E c3- A 1 3 R' T where r is the radius of the bubble. Similarly, for nitrogen: 4 - 7 r r 3 p N b n N b = Eq. A2 3 R' T In Equations A1 and A2, po D a n d PNb a r e t n e P a r t i a l pressures of oxygen and nitrogen in the bubble, R' is the universal gas constant and T is the absolute temperature. The total pressure of gases in the bubble, Pt, is given by: Pt = p o b + p N b + pH20 Eq. A3 where, pH20 is the vapor pressure of water at temperature T. Laplaces Equation defines the balance of pressure and surface tension forces acting on the bubble and is given by: 20 Pt = Pe + Eq. A4 r where Pe is the pressure in the liquid external to the bubble. For this derivation Pe is defined as: P e = pAtm. + P-g-h + Ps Eq. A5 where PAtm.> °> r> n> a r | d P s a r e t n e atmospheric pressure, the density of water, depth of fish in water column and system pressure where bubble growth is occurring, g is the gravitational constant. Equations A3 and A4 can be combined to give: 2 a POb + PNb + P H 2 ° = pAtm. + e-9* h + Ps + Eq. A6 r Solving for p^^ in Equation A6 and substituting the results in Equation A2 gives: n N b = ( P e r 3 + 2a r2 - p o b r " PH20 r ) E<3- A 7 3 -R' - T Equation A7 can be differentiated with respect to time to give: 222 dn Nb dt 4 • 7T 3-R'-T L ( 3-Pe-r 2 + 4-a-r - 3 - p o b - r 2 - 3 p H 2 0 - r ^ ) . d P 0 b dr dt - r3.. dt Eq. A8 Equation A1 can be differentiated with respect to time to give: d n o b 4 • 7T dt 3R' d p o b r 2 d r -, r 3 — + 3 p o b -dt dt Eq. A9 Equations A8 and A9 give the time rate of change in the number of moles of oxygen and nitrogen in the bubble. This change must be balanced by the rate of diffusion of these gases into the bubble. The diffusion equations for the movement of gases into the bubble can be written as follows. d n o b dt H 0 k 0 - 4 - 7 r r 2 - ( p 0 p - p o b ) Eq. A10 d nNb 0 = H N k N - 4 - 7 r r ^ - ( p N p - p N b ) Eq. A l l dt where, kg = mass transfer coefficient for oxygen kjsj = mass transfer coefficient for nitrogen POp = partial pressure of oxygen in plasma PNp = P a r t ' a ' pressure of nitrogen in plasma H Q = Henrys constant for oxygen HJSJ = Henrys constant for nitrogen 223 Using Equation A5 and A6, Equation A11 can be written as: dn Nb dt H N • k N • 4 . 7r • r . 2 • a PNp - Pe - + Pob + PH20 Eq. A12 Equating Equations A9 and A10 and Equations A8 and A12 gives two equations that can be combined into a single equation for the rate of change in bubble radius with time. The resulting equation is: R • T • dr dt W f c o p - P0b] + W 3Np" (Pe + V " P 0b " P H 2 ° ) (pe + - pH2ol ^ 3 T J Eq. A13 Nitrogen, in the vascular system of a fish, is biologically inert. Once gases in the fish are in a steady state relative to those in the water, p^p should be the same as the partial pressure of nitrogen in the environmental water (P|\j2)- T n u s > PNp = P N 2 - 0 n the other hand, oxygen is biologically active and in a state of constant transport from the water to the vascular system. Due to mass transport resistances at the gill membrane and utilization of oxygen by the gill, plasma dissolved oxygen partial pressure will be reduced from that of the water. Defining F as the ratio of the partial pressure of oxygen in the plasma to that of the environmental water, POp = F P ° 2 • 224 Finally, the Total Gas Pressure (TGP) in the environmental water is related to the component dissolved gases by: TGP = p02 + pN2 + pH20 Incorporating these expressions into Equation A13 yields: R ' -T • dr dt H 0 . k 0 - ( F p 0 2 - p o b ) • H N-k N-(pe 2 -a p Q b + p02 - TGPJ ( Pe + 4-o 3 T - pH20 ] Eq. A14 For a bubble that is not growing, dr/dt in the above equation is equal to zero. For bubble growth, dr/dt must be positive. Based on this growth criteria, the following criteria can be developed from Equation A14. For bubble growth: H 0 k 0 ( F P o 2 - p 0b) + % k N -2a Pe + p o b + p 0 2 - TGP > 0 or, 2a TGP > Pe + p 0 2 ( K F - 1) - (1 - K ) p o b Eq. A15 rO where K i s defined as: K = k N H N and r 0 is the initial radius of the bubble nucleus from which growth begins. Expanding Pe, Equation A15 becomes: 225 2 a TGP > P A t m + PS + ogh + p 0 2 ( K F - 1) - (1 - K ) p o b r o Eq. A16 Thus, Equation A16 is the general threshold equation for bubble growth in the vascular systems of fish exposed to supersaturated water. Because the swimbladders of physostome fish are highly vascularized with arterial blood, this organ can also grow in volume as a result of the transfer of supersaturated gases from the blood into the bladder. Assuming that the swimbladder of a fish acts like a very large bubble, Equation A16 can be used to define its threshold for overinflation. In this case, the nucleus radius, r0, is very large. As a result, the surface tension term, 2-a/r 0, is very small and can be neglected. Thus, the criteria for overinflation of the swimbladder becomes: TGP > PAtm. + Ps + o^g-h - p 0 2 ( K F - 1) - (1 - K ) p o b Eq.A17 Finally, for a bubble in the environmental water, the F term in Equation A16 is 1.0 and Ps = 0. Thus, for a bubble in the environmental water, the threshold criteria becomes: 2 a TGP > P A t m + p.g-h + + (K - l ) - ( P 0 b " P° 2) E (3' A 1 8 APPENDIX B TABLE I: SOURCES FOR WATER AND DORSAL AORTA p 0 2 DATA 226 AUTHOR CODE AUTHOR (S) 1 Cameron, J . C and Davis, J.C. (1970) 2 Eddy, F.B. (1977) 3 Kiceniuk, J.W. and Jones, D.J. (1977) 4 Stevens, E.D. and Randall, D.J. (1967) 5 Kiceniuk, J.W. (1969) 6 Stevens, E.D. (1967) 7 Holton, G.F. and Randall, D.J. (1967) 8 Thomas, S. and Hughes, G.M. (1982) 9 Sovio, A.; Nikinmaa, M.; Nyholm,K. and Westman, K. (1981) 10 Hobe, H.; Wood, C M . and Whetly, M.G. (1984) 11 Wood, C M . and Jackson, E.B. (1980) 12 Tuurala, H. (1983) 13 Smith, F.M. and Jones, D.J. (1982) 14 Spry, D.J. and Wood, C M . (1985) 15 Fidler, L.E. (1988) Complete references can be found in the bibliography of this thesis. TABLE II: LIST OF ABBREVIATIONS FOR TABLE III AUTHORS: Author code as shown in Table I WATER P 0 2 : p 0 2 of environmental water - mmHg. ARTERIAL P 0 2 : p 0 2 of arterial blood at dorsal aorta - mmHg. TEMP. O: Water temperature - deg. C. NO. SAMP.: Number of samples in measurement. RECORD NO: Record number assigned to data set. Std.Err.W.: Standard error in water p 0 2 measurement. Std.Err.A.: Standard error in blood p 0 2 measurement. F RATIO: Ratio of arterial p 0 2 to water p 0 2 . TABLE III: HATER AND DORSAL AORTA p02 DATA FROU THE LITERATURE FOR RAINBOU TROUT AUTHORS UATER P02 ARTERIAL P02 TEW. C. NO. SAHP. RECORD NO. Std. Err .U . Std. Err .A . F RATIO 1 155.000 133.200 1.001 0.S59 2 155.000 117.000 2.001 0.755 3 153.000 137.000 10.00 8 3.001 0.895 4 135.000 85.000 4.001 0.630 5 152.900 137.000 10.00 8 5.001 1.960 4.230 0.896 6 134.000 85.000 13 6.001 0.634 7 30.000 17.600 15.00 27 7.001 2.100 0.587 7 40.000 23.200 15.00 27 7.002 2.100 0.580 7 50.000 26.750 15.00 27 7.003 4.250 0.535 7 60.000 33.050 15.00 27 7.004 3.500 0.551 7 70.000 45.370 15.00 27 7.005 4.570 0.648 7 80.000 60.840 15.00 27 7.006 5.9S0 0.761 7 90.000 67.600 15.00 27 7.007 5.000 0.751 7 100.000 81.240 15.00 27 7.008 3.870 0.812 7 110.000 87.430 15.00 27 7.009 4.010 0.795 7 120.000 91.450 1S.00 27 7.01 5.640 0.762 7 130.000 105.710 15.00 27 7.011 3.020 0.813 7 140.000 111.200 15.00 27 7.012 4.290 0.794 7 150.000 121.340 7.013 8.090 0.809 8 155.000 108.000 15.00 6 8.001 3.000 11.000 0.697 8 60.000 23.000 15.00 6 8.002 3.000 6.000 0.383 8 155.000 115.000 15.00 8 8.003 4.000 10.000 0.742 9 155.000 125.730 9.00 9 9.001 6.190 0.811 9 73.580 68.310 9.00 9 9.002 6.540 0.928 9 73.5S0 63.240 9.00 9 9.003 3.630 0.859 9 73.580 61.890 9.00 9 9.004 9.450 0.841 9 155.000 117.150 9.00 9 9.005 4.000 0.756 9 155.000 120.600 9.00 9 9.006 4.360 0.778 9 155.000 116.300 9.00 9 9.007 5.300 0.750 9 155.000 120.630 9.00 9 9.008 2.500 0.778 9 155.000 115.550 9.00 9 9.009 2.900 0.745 10 150.400 105.600 13.00 12 10.001 0.702 10 496.000 376.000 13.00 12 10.002 0.758 10 544.000 356.800 13.00 12 10.003 0.656 10 528.000 329.600 13.00 12 10.004 0.624 10 560.000 360.000 13.00 10 10.005 0.643 11 162.100 111.000 15.00 7 11.001 14.730 9.820 0.685 11 407.660 314.300 15.00 7 11.002 12.300 29.460 0.771 11 419.450 315.300 15.00 7 11.003 13.750 18.600 0.752 11 402.750 335.000 15.00 5 11.004 9.820 23.600 0.832 11 429.270 336.900 15.00 5 11.005 12.280 19.640 0.785 11 471.500 324.160 15.00 5 11.006 14.730 14.730 0.688 11 399.800 349.700 15.00 4 11.007 12.230 22.100 0.875 12 153.040 113.280 10.00 12.001 0.740 00 rflBLE i l l : UATER AND DORSAL AORTA p02 DATA FROI1 THE L I T E R A T U R E FOR R A I N B O U TROUT AUTHORS UATER P02 ARTERIAL P02 TEHP. C. NO. SAHP. RECORD NO. Std. Err.U. Std. Err.A. F RATIO 12 14S.130 120.400 18.00 12.002 0.824 12 51.390 44.410 18.00 12.003 0.864 13 150.040 109.530 8.80 13.001 4.500 3.000 0.730 13 93.020 70.520 8.80 13.002 4.500 6.000 0.758 14 155.000 110.000 15.00 14.001 3.000 0.710 14 1S5.000 115.000 15.00 14.002 3.000 0.742 IS 15S.000 124.000 9.50 1 15.007 0.800 IS 155.000 111.000 9.50 1 15.008 0.716 IS 155.000 120.000 9.50 1 15.009 0.774 IS 155.000 117.000 9.50 1 15.01 0.75S 15 155.000 128.000 9.SO 1 15.011 0.826 IS 155.000 119.000 9.50 1 15.012 0.768 15 155.000 124.000 9.50 1 15.013 0.800 IS 155.000 117.000 9.50 1 15.014 0.755 15 155.000 122.000 9.50 1 15.015 - 0.787 IS 155.000 119.000 9.50 1 15.016 0.768 IS 155.000 115.000 9.50 1 15.017 0.742 15 155.000 119.000 9.50 1 15.018 0.768 IS 155.000 121.000 9.50 1 15.019 0.781 15 155.000 122.000 9.50 1 15.02 0.787 IS 155.000 119.000 9.50 1 15.021 0.768 15 155.000 123.000 9.50 1 15.022 0.794 15 155.000 121.000 9.50 1 15.023 0.781 15 155.000 117.000 9.50 1 15.024 0.755 15 155.000 124.000 9.50 1 15.025 0.800 15 155.000 122.000 9.50 1 15.026 0.787 15 155.000 109.000 9.50 1 15.027 0.703 15 155.000 119.000 9.50 1 15.028 0.768 15 1SS.000 117.000 9.50 1 15.029 0.755 15 155.000 118.000 9.SO 1 15.03 0.761 15 155.000 123.000 9.50 1 15.031 0.794 IS 155.000 120.000 9.50 1 15.032 0.774 IS 155.000 123.000 9.50 1 15.033 0.794 15 155.000 118.000 9.50 1 15.034 0.761 15 1S5.000 120.000 9.50 1 15.035 0.774 IS 1S5.000 125.000 9.50 1 15.036 0.806 15 155.000 118.000 9.50 1 0.761 IS 162.000 130.000 9.50 1 15.037 0.802 15 162.000 119.000 9.50 1 15.038 0.735 IS 162.000 128.000 9.50 1 15.039 0.790 15 162.000 123.000 9.50 1 15.04 0.7S9 IS 162.000 128.000 9.SO 1 15.041 0.790 15 162.000 122.000 9.50 1 15.042 0.753 15 183.000 147.000 9.50 1 15.043 0.803 15 183.000 133.000 9.50 1 15.044 0.727 15 183.000 140.000 9.50 1 15.045 0.765 IS 183.000 137.000 9.50 1 15.046 0.749 15 183.000 141.000 9.50 1 15.047 0.770 TABLE HI: HATER AND DORSAL AORTA p02 DATA FROH THE LITERATURE FOR RAINBOU TROUT AUTHORS HATER P02 ARTERIAL P02 TEHP. C. NO. SAMP. RECORD NO. Std. Err.U. Std. Err.A. F RATIO IS 175.000 137.000 9.50 1 0.783 15 175.000 126.000 9.50 1 0.720 IS 175.000 133.000 9.SO 1 0.760 IS 175.000 127.000 9.50 1 0.726 15 175.000 13S.000 9.S0 1 0.771 15 175.000 139.000 9.50 1 0.794 APPENDIX C: TABLE I: SOURCE OF DATA FOR GBT DATABASE Author Author(s) Code 1 Rucker (1975a) 2 Nebeker ef al. (1978) 3 Jensen(1980) 4 Ebel (1971) 5 Ebel (1969) 6 Rucker and Kangas (1974) 7 Ebel ef al. (1971) 8 Dawley et al. (1976) 9 Weitkamp (1976) 10 Wyatt and Beiningen (1969) 11 Blahm etal. (1974) 12 Meekin and Turner (1974) 13 Dawley and Ebel (1975) 14 Nebeker et al. (1979a) 15 Rucker (1975a) 16 Blahm etal. (1975) 17 Knittel etal. (1980) 18 Stroud and Nebeker (1976) 19 Nebeker et al. (1976a) 20 Coutant and Genoway (1968) 21 Nebeker etal. (1976b) 22 Nebeker and Brett (1976) 23 Nebeker etal. (1979) 24 Nebeker etal. (1980) TABLE II: LIST OF ABBREVIATIONS USED IN GBT DATABASE Record: Record number in database Author: Author data set identity number Species: Species Code; 1 = Chinook, 2 = Coho, 3 = Sockeye, 4 = Steelhead, 5 = Cutthroat. Stage: Stage Code; 0 = Eggs, 1 = Alevins, 2 = Fry, 3 = Adult Length: Fish length in mm. Weight: Fish weight in gr. Temp.C: Water temperature in deg. C. Patm.: Atmospheric pressure in mmHg. Depth: Water depth in M. %Mort.: Percent mortality. Time: Time to mortality in hrs. TGP%: Total Gas Pressure in percent of Patm. 02: Partial pressure of dissolved oxygen in mmHg. N2: Partail pressure of dissolved nitrogen in mmHg. pH20: Vapor pressure of water at Temp.C. Record Author Auth. No. Species Stage Length MM Ueight g TeHp. C 1 1.001 I ! 0 0 0 7.2 2 1.001 1 1 0 0 0 7.2 3 1.002 1 1 0 0 0 7.2 4 1.002 1 1 0 0 0 7.2 ct 1.003 1 1 0 0 0 7.2 6 1.003 1 1 0 0 0 7.2 7 1.003 1 1 0 0 0 7.2 8 1.003 1 1 0 0 0 7.2 9 2.001 2 4 1 35 0.3S 10 10 2.001 2 4 1 35 0.35 10 11 2.001 2 4 1 35 0.3S 10 12 2.001 2 4 1 35 0.35 10 13 2.001 2 4 1 35 0.3S 10 14 2.001 t. 4 1 35 0.35 10 15 2.001 1 i- 4 1 35 0.35 10 16 2.001 2 4 1 35 0.35 10 17 2.001 2 4 1 35 0.35 10 18 2.001 O 4 1 35 0.35 10 19 2.001 2 4 1 35 0.35 10 20 2.001 2 4 1 35 0.35 10 21 2.001 2 4 1 35 0.35 10 22 2.001 2 4 1 35 0.35 10 23 2.001 2 4 1 35 0.35 10 24 2.001 2 4 1 35 0.3S 10 25 2.001 2 4 1 35 0.3S 10 26 2.002 2 4 1 35 0.35 10 27 2.002 2 4 1 35 0.35 10 23 2.002 2 4 1 35 0.35 10 29 2.002 i. 4 1 35 0.35 10 30 2.002 2 4 1 35 0.35 10 31 2.002 2 4 1 35 0.35 10 j2 2.002 2 4 1 35 0.35 10 33 2.002 2 4 1 35 0.35 10 31 2.002 2 4 1 35 0.35 10 35 2.002 2 4 1 35 0.35 10 36 2.002 2 4 1 35 0.35 10 37 2.002 2 4 1 35 0.35 10 38 2.002 2 4 1 35 0.35 10 39 2.002 2 4 1 35 0.35 10 40 2.002 2 4 1 35 0.35 10 41 2.003 2 4 1 35 0.35 10 42 2.003 2 4 1 35 0.35 10 43 2.003 2 4 1 35 0.35 10 44 2.003 2 4 1 35 0.35 10 45 2.003 2 4 1 35 0.3S 10 46 2.003 2 4 1 35 0.35 10 47 2.003 ••> c 4 1 35 0.35 10 43 2.003 O 4 1 35 0.35 10 49 2.003 2 4 1 35 0.35 10 50 2.003 2 4 1 35 0.35 10 51 2.003 2 4 1 35 0.35 10 2.003 2 4 1 35 0.35 10 iHHg Depth M Z Hort. Tine hr TOP Z 02 HMHg N2 MuHg pH20 H M H Q 758 0 11 1032 93 149 63 592.88 7.617 753 0 19 1560 99 149 63 532.33 7.617 758 0 1.4 1560 109.7 154 35 66S.96 7.617 753 0 1.3 1032 109.7 154 35 663.96 7.617 758 0 0.6 1032 113.6 162 23 729.24 7.617 758 0 0.4 1032 113.6 162 .23 729.24 7.617 758 0 0.1 1560 118.6 162 23 729.24 7.617 758 0 0.1 1560 118.6 162 23 729.24 7.617 754 0.08 60 600 126.2 0 0 9.208 754 0.08 90 1080 126.2 n 0 9.208 754 0.08 60 600 126.2 0 0 9.203 754 0.08 50 552 126.2 0 0 9.208 754 0.08 84 840 126.2 n 0 9.203 754 0.08 70 648 126.2 0 o 9.208 754 0.08 90 1080 126.2 0 0 9.208 754 0.08 10 432 126.2 0 0 9.203 754 0.08 78 720 126.2 0 0 9.208 754 0.08 25 492 126.2 0 0 9.208 754 0.08 20 430 126.2 0 n 9.203 754 0.03 18 430 126.2 0 0 9.208 754 0.08 30 504 126.2 0 0 9.208 754 0.08 88 960 126.2 n o 9.208 754 O.OS 93 1200 126.2 0 0 9.208 754 0.08 40 528 126.2 0 0 9.208 754 0.08 80 756 126.2 0 0 9.208 754 0.08 10 480 122.9 0 0 9.203 754 0.08 50 672 122.9 0 0 9.208 754 0.08 20 504 122.3 0 n 9.203 754 0.08 10 430 122.9 0 0 9.208 754 0.08 63 840 122.9 0 0 9.208 754 0.08 55 720 122.9 n n 9.208 754 0.08 40 612 122.9 n o 9.203 754 O.OS 60 763 122.9 0 0 9.208 754 0.08 38 600 122.9 n n 9.208 754 0.08 70 1056 122.3 0 0 9.208 754 0.08 68 960 122.9 0 0 9.208 754 O.OS 30 552 122.9 n 0 9.208 754 0.08 74 1200 122.9 0 0 9.203 754 0.08 25 528 122.9 0 0 9.208 754 0.08 71 1080 122.3 0 0 9.208 754 O.OS 63 1200 113.4 0 0 9.208 754 0.03 30 864 113.4 0 0 9.208 754 0.08 30 840- 113.4 0 0 9.208 754 O.OS 7.5 600 113.4 0 0 9.203 754 O.OS 20 720 113.4 0 0 9.203 754 O.OS 40 972 113.4 0 0 9.203 754 O.OS 20 720 113.4 0 0 9.203 754 0.08 25 792 118.4 0 0 9.203 754 O.OS 10 612 113.4 0 0 9.203 754 0.08 50 1032 113.4 0 0 9.203 754 O.OS 38 960 113.4 0 0 9.208 754 O.OS 1080 113.4 0 o 9.203 234 co co co co o:> o:- & co co co co co co co co o:> r- r- r- r-_ u'i T T T *r T V rr CM CM CM CM CM fw r_ u-t U-I U-I U"I LTI U-I u"i co co or- co in i .3. .—• c:- o o o 0 0 o o • o o o o —1 u"i u-i LTI O O-J CM c-j -."M CM *M CM CM r— r— r— r— r— u-i u't o o •:=• o 0 0 o <r- T T -•.••••j CM CM CM oj CM CM CM CM CM CM CM CM '--.J CM CM CM LTI -X* ' X - -x- m <x- ix- 'X> «X> LO ' X - -x< ix- r~ r-- r- r- f- X - -X- in ro m in m in in co U'I u"i u* u"> 1 cr. <r* cr- tr" cr- cr" cr< en cr. tr- cr. cr. c< -T- cr o-< cr> © o o o T4 C-J CM CM CM CM CM CM CM U'" 1/ ix" U -* IX* © O *-« * H rt rt ,v" rn • , o © O O © © O O O O O O CC' !••- f- r- iT< U'I LTI U'l U'i LA U'l Lfi LI 0 : > » CO N T T V "T T T ' ffi O O O 7 ff-! fO fH n m (i'i uS y5 O O T T T T T V 7 U*i U1 U"i LTi T T U*( U"l LCl LTi IXl U1 LCi U'i l-Tt U'l LTi LT) U"i U ^ V tr U"l LA LTi LTi U"i U'I U'l CC' CO CC' x- r- f- r- r-- r- r- r- rw r-_ r_ r-- r- jv- r- r- r^ - r- r- r - r- r - r- r - rs_ r- -J:- >X' O:< r- r^ - r--• o o o o o c< o o 0 0 0 0 cr. • © c- c o o T T T T T T T T T T 7 W CT' w r i TT rn o o o o o o o c:- U'I u"i u"i U'I U". U'I u". u"i u*i w> O-J ui' CM U"i co o:> u-i H H H H H H H H H H Oi fsl ^ -H H t-l H ' o o c- o c- c- o c< c- o o © o o o o o o o o 1 o © ©i © ©' © © © © 1 © © © © © >X> O © CC' CM © CC' © © CO CM CM CM CM CM CM CM LO CO CO T CO T CO 0!" CO T CC' 0j CC' "T "T CC' "T CC' CO CC' OS CC' T CO CO TT 0? f-— O © O © ©« © CM ^ CM © V Oi C/> CO N- f - r- N- r - IS- • >_C- T CM 'X' CM *T T u3 0-J 'J^ uT' 'X' CM 0J uT' CM LC1 'X1 w T CJ V uT- 0J -X1 • CO W W T T T r - *H o CM CM © O CO CC' CM CM CM CM CM CM CM C"' •>-< —' *H rt rt rt „ w rt rt <r, ^ ,r..j w fsj ( > J ,7^  rt rt rt rt rt rt rt rt rt rt rt T-H rt C rt i,*-J CJ CM 0 0 0 0 © 0 0 © © 0 © O O C ' © © © © © © © 0 I LA U'i U'l U'i U'i U'i U'i LTi U'I • r - r - r - r - r - r - r - • r-~ T • © © © © © © ' - t © C--1 U'i LTi ' X - (n U'i m <x> © " T T T T T ""sT *T V V "T T T^" V ^ kX' 0"' CTi CT> \T> <T" C' CT' 0"' 0*" CT' tT^  tr"1 tT' Cn 0~» sT"' tT> <?"' <T"i C ' 0*^  C"» 'T' 0^ CT' C ' C^ CT1 U"i U'i U ^ U'i U'l U"i LTi LTI U"> LTI U'I LTI LTI U'I U'I U'I U'I U1 •£> T T T T T T T T ' T TT T T T T T T TT T TT " T T T T fs_ r^ - r^ - r^ - rw r - rs- r^ - rs-O 0 © c © 0 © © © © © © © • 0 © © © © CM 0 —H rt rt rt rt rt rt rt rt rt rt a. r a-1— U'i LTI Wl LTi U'l U-! U'l U'i LTi in T CM T LTi CM m 0-1 in n-i m in in m © C' © © 0 © 0 © © © © © © © © * X L0 LTi U~i tXi IXt LTi U'i m CO m T TT r m in f i m in | V | m m in X' r~- U'i in 0-1 CM in f'-| in in 0J 4-' Cn C * ' » K> K ' n m n f>-i n w M H rt H • rt -rt r^t fT| C1"' C> 0 -' CM CM CM in T T T T T ^ T T co co co co CM CM in m o-i »n m in in x- u*i u'i u*i ' © © © © o © © o © © o © © © © © © © © © © © > © © © © 0 0 © o \ C^J CM CM CM CM CM CM CM CM CM CM CM Osl CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CMCM'NCMCM":M<\ l0dOJ<\ lCM0JCMeM'N<NJ0J^in n 7 V T T T T V V ' T V U"l C C " - i H f sj ii", © © © © © © © © © © © © © C ' - ^ ' - ' r t © © © © • © © C' © © © © © © • O © © ^ rt rt rt rt rn -^ r TT -r u*i N - o:> tT< <T' - " © • C O © © © © © © © © © © © © © © © © o © © © © © © © © o © © © © 1 CM in T T b"l U'i U'l X> I"1- rt r>-, tr U'i f-1 rt rt rt rt rt rt rt rt rt rt rt F~I O © O O © ' © © © © © © © © © © © © © © © © © CM CM CM CM CM Od CM CJ CM CM CM CsJ ' N i r ^ ' r j ^ n T T ' T ^ T V T r T ' T V T T ' n - T T T T V T V T T T T T T l/i U'i L/i U" U'i U"i d Author fiuth. Ho. Species Stage Length MM Ueight g Te«p. C F'atM MdHg Depth H hort. TiHe hr TOP •/. 02 MMHg N2 tmHg pH20 HMHg 105 5.008 5 2 2 0 0 0 749 70 240 0 0 823.4"' 4.58? 106 5.009 5 2 2 0 0 0 749 1.5 100 240 0 0 323.43 4.533 10? 5.01 5 2 2 0 0 0 749 3.5 240 0 0 823.43 4.583 108 5.011 5 2 0 0 0 749 6 18 240 0 0 323.43 4.583 109 6.001 6 1 1 0 0 10 760 0 6 1320 112 176.5 664.33 9.208 110 6.001 6 1 1 0 0 10 760 0 20 -132 112 176.5 664.38 9.208 111 6.001 6 1 1 0 0 10 760 0 25 -132 112 176.5 664.38 9.203 112 6.001 6 1 1 0 n 10 760 0 5 1030 112 176.5 664.33 9.208 113 6.002 6 1 1 0 0 10 760 0 C 1010 116 182.31 638.11 9.208 114 6.002 6 1 1 0 n 10 760 n 10 1320 116 182.31 688.11 9.208 115 6.002 6 1 1 0 0 10 760 0 20 -132 116 182.81 688.11 9.203 116 6.002 6 1 1 0 0 10 760 0 25 -132 116 132.81 688.11 9.208 117 6.003 6 1 1 0 0 10 760 0 25 817.5 120 189.11 711.84 9.208 118 6.003 f. 1 1 0 0 10 760 0 20 735 120 133.11 711.84 9.208 119 6.003 6 1 1 0 0 10 760 0 SO 1070 120 189.11 711.84 9.208 120 6.003 6 1 1 0 0 10 760 0 10 725 120 133.11 711.84 9.203 121 6.003 6 1 1 0 n 10 760 0 30 350 120 139.11 711.34 9.208 122 6.003 6 1 1 0 0 10 760 0 53 1320 120 189.11 711.84 9.203 123 6.003 8 1 1 0 0 10 760 0 40 970 120 139.11 711.34 9.208 124 6.003 6 1 1 0 0 10 760 0 c; 640 120 139.11 711.84 9.203 125 6.004 6 1 1 0 0 10 760 0 5 550 124 195.41 735.57 9.208 126 6.004 6 1 1 0 n 10 760 0 60 1050 124 195.41 735.57 9.203 127 6.004 8 1 1 0 0 10 760 0 30 720 124 195.41 735.57 9.203 128 6.004 6 1 1 0 0 10 760 0 40 300 124 195.41 735.57 9.208 129 6.004 6 1 1 0 0 10 760 0 20 660 124 195.41 735.57 9.208 130 6.004 6 1 1 0 0 10 760 0 10 610 124 195.41 735.57 9.208 131 6.004 6 1 1 0 0 10 760 0 50 910 124 135.41 735.57 9.203 132 6.004 6 1 1 0 0 10 760 0 25 690 124 195.4) 735.57 9.203 133 6.004 6 1 1 0 0 10 760 0 70 1320 124 135.41 735.57 9.203 134 6.005 6 1 1 0 0 10. 760 0 70 940 128 201.72 759.3 9.203 135 6.005 6 1 1 0 0 10 760 0 10 600 123 201.72 759.3 9.208 136 6.005 6 1 1 0 0 10 760 0 50 315 128 201.72 759.3 9.203 137 6.005 6 1 1 0 0 10 760 0 20 650 123 201.72 759.3 9.208 138 6.005 8 1 1 0 0 10 760 0 40 760 123 201.72 759.3 9.203 139 6.005 6 1 1 0 0 10 760 0 -•>ct 630 128 201.72 759.3 9.203 140 6.005 6 1 1 0 0 10 760 0 5 520 128 201.72 759.3 9.208 141 6.005 6 1 1 0 0 10 760 0 1 O 1320 128 201.72 759.3 9.203 142 6.005 6 1 1 0 0 10 760 0 30 710 128 201.72 759.3 9.208 143 6.005 6 1 1 0 0 10 760 0 60 870 128 201.72 759.3 9.208 144 6.006 6 1 1 0 0 8 760 0 25 -204 111 157.84 677.3 8.045 145 6.006 6 1 1 0 0 8 760 0 10 -204 111 157.84 677.3 3.045 146 6.006 6 1 1 0 0 8 760 0 20 -204 111 157.84 677.3 8.045 147 6.007 6 1 1 0 0 8 760 o 10 -204 111 157.84 639.18 8.045 148 6.007 6 1 1 0 0 8 760 0 20 -204 112 157.34 683.18 3.045 149 6.007 6 1 1 0 n $ 760 0 or -204 112 157.84 689.18 3.045 150 6.008 6 1 1 0 n R 760 0 25 -204 114 159.41 701.06 8.045 151 6.008 f. 1 1 0 0 y 760 0 20 1360 114 153.41 701.06 3.045 152 6.008 6 1 1 0 0 8 760 0 t-2. 2040 114 159.41 701.06 3.045 153 6.008 & 1 1 0 n 8 760 n 10 1730 114 159.41 rot. Of. 8.045 154 6.009 6 1 1 0 0 ft 760 0 30 1550 116 160.99 712.34 8.045 155 6.009 6 1 1 0 0 8 760 0 25 1450 116 160.99 712.94 8.045 156 6.009 f. 1 1 0 n o 760 n 10 1130 116 160.99 712.94 8.045 Record Author Auth. Ho. Species Stage Length HH Height g TeHp. C PatM HtiHg Depth M flort. TiHe hr TGP Z 02 miHg H2 HHHQ pH20 MhHq 157 6.009 6 1 1 0 0 3 760 0 55 2040 116 160.99 712.94 8.045 158 6.009 6 1 1 0 0 r. 760 0 40 1770 116 160.99 712.94 8.045 159 6.009 6 1 1 0 0 8 760 0 20 1350 116 160.99 712.94 8.045 160 6.009 6 1 1 0 0 8 760 0 50 1360 116 160.39 712.94 8.045 161 6.01 6 1 1 0 0 8 760 0 20 390 118 162.57 724.33 8.045 162 6.01 6 1 1 0 0 8 760 0 50 1080 118 162.57 724.33 8.045 163 6.01 6 1 1 0 0 3 760 n 80 1350 118 162.57 724.33 8.045 164 6.01 6 1 1 0 0 3 760 0 10 800 118 162.5? 724.33 8.045 165 6.01 6 1 1 0 0 8 760 0 60 1110 113 162.57 724.83 8.045 166 6.01 6 1 1 0 0 Q 760 n 25 925 113 162.5? 724.83 8.045 167 6.01 6 1 1 0 0 3 760 0 30 960 118 162.57 724.33 8.045 168 6.01 6 1 1 0 0 8 760 0 40 1020 IIS 182.57 724.83 8.045 169 6.01 6 1 1 0 0 8 760 0 70 1150 113 162.57 724.83 8.045 170 6.01 6 1 1 0 0 8 760 o 90 2040 u s 162.57 724.83 8.045 171 6.011 6 2 1 0 0 8 760 0 10 -204 i n 157.84 677.3 8.045 172 6.011 6 2 1 0 0 8 760 0 25 -204 i n 157.34 677.3 8.045 173 6.011 6 2 1 0 0 8 760 0 20 -204 i n 157.84 677.3 8.045 174 6.012 6 2 1 0 0 8 760 0 10 -204 112 157.34 689.18 8.045 175 6.012 6 2 1 0 0 8 760 0 25 -204 112 157.84 689.18 8.045 176 6.012 6 2 1 0 0 3 760 0 20 -204 112 157.34 689.18 8.045 177 6.013 6 2 1 0 0 8 760 0 or -204 114 159.41 701.06 8.045 178 6.013 6 2 1 n 0 3 760 0 20 2040 114 159.41 701.06 8.045 179 6.013 6 2 1 0 0 3 760 0 10 1250 114 159.41 701.06 8.045 180 6.014 6 2 1 0 0 8 760 0 20 1560 116 160.99 712.94 8.045 181 6.014 6 2 1 0 0 8 760 0 10 1030 116 160.99 712.94 8.045 182 6.014 8 2 ] 0 0 3 760 0 30 1920 116 160.99 712.94 8.045 183 6.014 6 2 1 0 0 8 760 0 35 2040 116 160.99 712.94 8.045 184 6.014 6 2 1 0 0 3 760 0 25 1740 116 160.99 712.94 8.045 185 8.015 6 2 1 0 0 A O 760 0 40 950 US 162.57 724.33 8.045 186 6.015 6 2 1 0 0 8. 760 0 50 11S0 118 162.57 724.33 8.045 187 6.015 6 2 1 0 0 8 760 0 30 870 118 162.57 724.33 8.045 188 6.015 6 2 1 0 0 8 760 0 60 1360 118 162.57 724.83 8.045 189 6.015 6 2 1 0 0 3 . 760 0 10 660 118 162.57 724.33 8.045 190 6.015 6 0 0 8 760 0 70 1600 113 162.57 724.33 8.045 191 6.015 f. 2 1 0 0 8 760 0 87 2040 118 162.57 724.33 8.045 192 6.015 6 2 1 0 0 3 760 0 20 760 118 162.57 724.83 8.045 193 8.015 6 2 1 0 0 8 760 0 25 815 118 162.5? 724.33 8.045 194 6.015 8 2 1 0 0 y 760 n 30 1310 113 162.57 724.33 8.045 195 6.016 6 2 1 0 0 8 760 0 10 750 115 157.34 70? 8.045 196 6.016 6 2 1 0 0 3 760 0 14 1008 115 157.34 707 8.045 137 6.016 8 2 1 0 0 760 0 20 -100.8 115 157.34 70? 8.045 198 6.016 6 2 1 0 0 P 760 0 25 -100.8 115 157.84 707 8.045 199 6.017 6 2 1 0 0 8 760 0 40 680 117 160.39 724.83 8.045 200 6.017 6 2 1 0 0 S 760 0 30 515 117 160.39 724.33 8.045 201 6.017 6 2 1 0 0 8 760 0 10 250 117 160.99 724.83 3.045 202 6.017 6 2 1 0 0 8 760 0 25 452.5 118 160.99 724.83 3.045 203 6.017 6 z 1 0 n 8 760 0 50 1008 117 160.99 724.33 8.045 204 6.017 6 2 1 0 0 8 760 0 20 330 117 160.99 724.3? 8.045 205 6.018 8 2 1 0 0 8 760 o 10 120 121 162.57 748.53 8.045 206 6.013 8 2 1 0 0 S 760 0 30 275 121 162.57 748.59 3.045 207 6.018 6 2 1 0 0 3 760 0 70 300 121 162.57 748.59 8.045 203 6.018 6 2 1 0 0 R 760 0 20 200 121 162.57 743.59 8.045 Record Author Ruth. Mo. Species Stage Length MM Ueight g Te«p. C PatM MMHg Depth H V. Hort. Tine hr TGP '/. 02 «MHg N2 MMHg pH20 MMHg 209 6.018 6 2 1 0 0 8 760 0 60 570 121 162.57 748.59 8.045 210 6.018 6 2 1 0 0 8 760 0 40 360 121 162.57 748.59 8.045 211 6.018 6 2 1 0 0 8 760 0 50 450 121 162.5? 748.59 8.045 212 6.018 6 2 1 0 0 8 760 0 74 1008 121 162.5? 748.59 8.045 213 6.018 6 2 1 0 0 8 760 0 or 237.5 1.21 162.5? 748.59 8.045 214 7.006 7 1 2 134 23 15 760 0.2 50 11.3 0 0 752.73 12.737 215 7.006 7 1 2 134 23 15 760 0.2 15 8.5 0 0 752.73 12.737 216 7.006 7 1 2 134 23 15 760 0.2 c 3.5 o 0 752.73 12.737 217 7.006 7 1 2 134 23 IS 780 0.2 100 15 0 0 752.73 12.787 218 7.007 7 1 2 134 23 20 760 0.2 100 13 0 0 747.94 17.534 219 7.00? 7 1 2 134 23 20 760 0.2 5 4.3 0 0 747.94 17.534 220 7.00? 7 1 2 134 23 20 760 0.2 15 6 0 0 747.94 17.534 221 7.007 7 1 2 134 23 20 760 0.2 50 7.5 0 0 747.94 17.534 222 7.008 7 1 2 134 23 23 760 0.2 5 2.5 0 0 744.38 21.069 223 7.008 7 1 2 134 23 23 780 0.2 15 5.8 0 0 744.38 21.069 224 7.008 7 1 2 134 23 23 760 0.2 50 7.3 0 n 744.38 21.069 225 7.008 ? 1 2 134 23 23 760 0.2 100 11.5 0 0 744.33 21.069 226 7.014 7 2 2 122.7 19.1 23 760 0.2 c 9.3 0 0 744.33 21.069 227 7.014 ? 2 2 122.7 19.1 23 760 0.2 100 17.3 0 0 744.38 21.069 228 7.014 7 2 2 122.7 19.1 23 760 0.2 15 3.5 o 0 744.38 21.069 229 7.014 7 2 2 122.7 19.1 23 760 0.2 50 13 0 0 744.38 21.069 230 7.015 7 2 2 122.7 19.1 20 760 0.2 100 15.3 0 n 747.94 17.534 231 7.015 7 2 2 122.7 19.1 20 760 0.2 15 3.3 0 n 747.94 17.534 232 7.015 7 2 2 122.7 19.1 20 760 0.2 50 13.5 0 0 747.94 17.534 233 7.015 7 2 2 122.7 19.1 20 760 0.2 5 6.3 n 0 747.94 17.534 234 7.016 7 2 2 122.7 19.1 15 760 0.2 50 3.3 n 0 752.73 12.78? 235 7.016 7 2 2 122.7 19.1 15 760 0.2 50 18.1 0 0 752.73 12.737 236 7.016 7 2 2 122.7 19.1 15 760 0.2 15 7.9 0 0 752.73 12.737 237 7.016 7 2 o 122.7 19.1 IS 760 0.2 6.9 0 fl 752.73 12.73? 238 7.016 7 2 2 122.7 19.1 IS 780 0.2 5 7 0 0 752.73 12.78? 239 7.016 7 2 2 122.7 19.1 15 760 0.2 15 7.2 0 0 752.73 12.78? 240 7.017 7 c 2 122.7 19.1 10 760 0.2 cr 12.9 0 0 756.33 9.208 241 7.017 7 2 2 122.7 19.1 10 760 0.2 15 14.6 0 0 756.33 9.208 242 7.01? 7 2 2 122.7 19.1 10 760 0.2 100 16.5 n 0 756.33 9.208 243 7.01? 7 2 2 122.7 19.1 10 760 0.2 c 6.9 0 0 756.33 9.203 244 7.017 7 2 2 122.7 19.1 10 760 0.2 50 10 0 0 756.33 9.208 245 7.017 7 2 2 122.7 19.1 10 760 0.2 15 7.6 0 0 756.33 9.208 246 7.018 7 2 2 122.7 19.1 5 760 0.2 15 10.3 0 n 759.02 6.543 247 7.018 7 2 2 122.7 19.1 5 760 0.2 5 8.8 0 0 759.02 6.543 248 7.013 7 2 2 122.7 19.1 5 760 0.2 50 13.5 n 0 759.02 6.543 249 7.02 7 1 2 123 19 10 760 0.2 5 10 0 0 756.33 9.208 250 7.028 7 4 2 179 54 15 760 0.2 100 11 0 0 752.73 12.787 251 7.028 7 4 2 173 54 15 760 0.2 50 14 0 0 752.73 12 > 757 252 7.028 7 4 2 179 54 15 760 0.2 50 5.7 fl 0 752.73 12.78? 253 7.028 7 4 2 179 54 IS 760 0.2 100 22 0 0 752.73 12.737 254 7.028 7 4 2 179 54 15 760 0.2 15 3.6 0 0 752.73 12.73? 255 7.028 7 4 2 179 54 15 760 0.2 15 10.8 0 0 752.73 12.787 256 7.023 7 4 179 54 15 760 0.2 c 2.9 0 0 752.73 12.737 257 7.028 7 4 2 179 54 15 760 0.2 c; 9.5 0 n 752.73 12.737 to 258 7.03 ? 4 179 54 10 760 0.2 5 3.3 0 0 756.33 9.203 W 259 7.03 7 4 2 179 54 10 760 0.2 15 4.7 0 0 756.33 9.208 ^ 260 7.03 i' 4 2 179 54 10 760 0.2 100 11 0 0 756.33 9.208 fluth. No. Species Stage Length MM Ueight g TeMp. C PatM MMHg Depth M V. Hort. TiMe hr TGP Z 02 MMHg N2 MMHg pH20 MMHg 261 7.03 7 1 1 C 179 54 10 262 3.001 8 X 2 130 54.3 10 263 8.003 8 z 40 0.4 10 264 8.004 8 I 2 40 0.4 10 265 3.005 8 2 40 0.4 10 266 8.007 8 * 2 180 54.8 10 267 8.013 8 1 2 40 0.4 10 268 8.014 8 I 2 40 0.4 10 269 8.015 8 I 2 40 0.4 10 270 8.016 8 I 2 40 0.4 10 271 8.017 8 I 2 40 0.4 10 272 8.018 8 L 2 40 0.4 10 273 8.019 8 I 2 40 0.4 10 274 8.02 8 I 2 40 0.4 10 275 8.021 8 I 2 40 0.4 10 276 8.022 8 2 40 0.4 10 277 8.023 8 I 2 40 0.4 10 278 8.024 8 I 2 40 0.4 10 279 8.025 8 1 2 180 54.8 10 280 8.026 8 ' 1 2 180 54.8 10 281 8.027 8 1 2 180 54.8 10 282 8.028 8 X 2 180 54.3 10 283 3.029 S X 2 180 54.8 10 284 8.03 8 X 2 180 54.8 10 285 8.031 8 X 2 180 54.8 10 236 8.032 8 I 2 180 54.8 10 287 8.033 8 \ 2 180 . 54.8 10 238 8.034 8 \ 2 180 54.3 10 289 8.035 8 ' I 2 180 54.8 10 290 3.036 8 1 2 180 54.8 10 291 9.001 9 I 2 0 0 0 292 9.001 9 I 2 0 0 0 293 9.002 9 0 0 0 294 9.002 9 2 0 0 0 295 9.003 9 I 2 0 0 0 296 9.004 9 I 2 0 0 0 297 9.005 9 I 2 0 0 0 298 9.006 9 I 2 0 0 o 299 9.007 9 1 2 0 0 0 300 9.03 9 1 2 0 0 0 301 10.001 10 1 2 0 0 0 302 11.001 11 I 2 0 0 0 303 11.002 11 2 0 0 0 304 11.003 11 < 1 2 n 0 0 305 11.004 11 ' 1 2 0 0 0 306 12.001 12 I 1 0 0 11.1 307 12.001 12 I 1 0 0 11.1 308 12.001 12 1 1 0 0 11.1 309 12.001 12 ' X 0 0 0 11.1 310 12.001 12 I 1 0 n 11.1 311 12.001 12 ' X 0 0 0 11.1 312 12.001 12 I 1 n 0 11.1 760 0.2 50 5.1 0 0 756.33 9.203 760 0.25 57 168 115.4 132.02 686.33 9.203 760 0.25 30 1440 115.4 132.02 686.33 9.203 760 0.25 97 1440 120.1 191.79 711.34 9.208 760 0.25 18 1440 109.4 170.2 652.52 9.208 760 0.25 100 48 122.2 210.78 708.37 9.203 760 0.25 20 168 120.1 191.79 711.84 9.208 760 0.25 25 216 120.1 191.79 711.84 9.203 760 0.25 50 480 120.1 191.79 711.84 9.203 760 0.25 70 672 120.1 191.79 711.34 9.208 760 0.25 20 432 115.4 182.02 636.33 9.203 760 0.25 25 624 115.4 182.02 686.33 9.208 760 0.25 50 1030 115.4 132.02 686.33 9.203 760 0.25 70 1272 115.4 182.02 686.33 9.203 760 0.25 20 -144 109.4 170.2 652.52 9.208 760 0.25 25 -144 109.4 170.2 652.52 9.203 760 0.25 50 -144 109.4 170.2 652.52 9.208 760 0.25 70 -144 109.4 170.2 652.52 9.208 760 0.25 20 18 122.2 210.73 708.87 9.203 780 0.25 25 21.6 122.2 210.78 708.87 9.203 760 0.25 50 28.8 10O "* LI-C » £. 210.78 708.87 9.208 760 0.25 70 31.2 122.2 210.78 708.87 9.208 760 0.25 20 66 115.3 189.42 677.44 9.203 760 0.25 25 69.6 115.3 189.42 677.44 9.208 760 0.25 50 112.3 115.3 189.42 677.44 9.208 760 0.25 70 -16.8 115.3 139.42 677.44 3.203 760 0.25 20 -16.8 103.5 170.83 652.52 9.208 760 0.25 25 -16.8 103.5 170.33 652.52 9.203 760 0.25 50 -16.8 109.5 170.33 652.52 9.203 760 0.25 70 -16.8 109.5 170.33 652.52 9.208 743 4 20 -144 121 0 0 4.583 743 4 0 -144 121 0 0 4.583 743 3 0 -144 121 0 0 4.583 743 3 20 -24 121 0 0 4.533 743 4 0 -144 124 0 0 4.533 743 3 20 -48 124 0 0 4.583 743 0.1 53 240 121 0 o 4.533 743 0.25 50 48 125 0 o 4.533 743 0.25 50 240 120.5 0 0 4.583 743 4 20 -48 124 0 0 4.533 0 0.6 100 C; 0 0 -5.5 4.533 759 2.5 11 1320 0 0 718.26 4.533 759 1 80 1320 0 0 718.26 4.533 759 2.5 6 1320 0 0 718.26 4.583 759 1 80 1320 0 0 718.26 4.533 736 0.17 25 2316 111.8 112.73 699.89 9.903 736 0.17 40 2424 111.3 112.73 699.39 9.909 736 0.17 30 2400 111.8 112.78 699.89 9.909 736 0.17 54 792 111.8 112.73 699.39 9.903 736 0.17 10 2136 111.8 112.78 699.89 9.909 736 0.17 77.3 648 111.8 112.73 699.89 9.909 736 0.17 60 2520 111.8 112.73 639.89 9.309 239 C". tf-. tf-- tf"> tf"-£Pi tf*. Cn tf"> tf-' <T" tf"> tf"1 0~* tf"" tf"1 o o o o o o o o o o O Cn tf-. tf-. .T- tf". tf-. cn tf". tf% .3-' tf-' <T> tf"i <T< i^ Ti tf"i ITI ITI tf"i tf"i o o o o o o o o c 'T> tf". "T- tf"- C"- 0~- <T< tf"< tf"* tf\ <T- tf". tf"' <T< tf-1 tf". O O O O O O O c . tf-i tf-. cr, cr» tf-- C' tf*. tf". iT*. .7-1 tf-. tf-. tf-. tf-. tf-. tf-i cr. c""i tf". iT- O tf". tf". >T> C"' O O O O O O O O O O O O O O O O O O o o "T" CT' 'T. tf"> tf"> tf". iT> tf"» tf"- CT> tf". tf". tf-» »T' tf"> tf"> tf"i tf". tf". tf"> tf-. tf" tf"- CT• tf*> <T> T- tf"> tf"i tf*> C- <Tr tf*> 0"' tf". tf-' tf"> tf"' tf"' tf*» CT- <?< tf"' <T' tf". <T< tf"- tf*' tf"- tf"> C- 0". tf". tf". tf"> tf"' tf". tf". tf*> tf"- tf"i tf". tf". tf". tf"' tf". tf". tf". tf-. tf", tf-, > 0"| CO CO CO CO CO C- C^ . > o*- co co co a- o:> cr- c< 1 'X* -X- TO TO -X- TO TO TO • co co co co co co co co co co co co 00 o:-1 co co co co co q'j co co co e:> co co co co • TO TO TO TO X- TO TO TO TO TO TO TO TO 'X' TO TO X' TO< TO< •T- CT. tf"< tf"' tf". o*. - u_> TO . o-> tf-- tf-, tf-. cr. 0*^  cr. cr. cr. v< 1- r - r-- r - t- r -. CT. tf". tf". tf". tf". tf". tf". CP tf". .Ti —I ' T-I i-t i-t *-< -*. > 'X> x> TO TO TO X> TO TO X- TO .•— r-- r*- r-- r - r- iw _  _ _ _ _ _ _ _ _ _ _ __ __ __ _ _ __ _______ 'X' _• 'X- TO TO . f- r - m tn tn tn -n i n tn i n in in tn in m in 0*. in r - r - r - r— r - r- r - r - r-- r - h- r - r - r - r - N- r - cj CJ e J OJ CJ OJ eg eg c-_ CJ r- r-- r-. r_. r_. csj c-j CJ r-- r - r - r - r-- r-- r - r-- r - r^ - r - r - r - r - p- r - 0^  cj cj CJ CJ C-J C-J CJ CJ OJ CJ CJ CJ CJ fsj eg I?J H H H »H J H H co o:' a ' « ' 0 o o o ' 00 CO CC' ' c" o o - co o:> co cc> • o o o c" . CO CO 0J CO CC' ' O C' o o o > o:« CC' TO co TO co o:' cc< co TO CO CO O:> TO CO O? CO . co T 7 T T 7 7 1 Csi C\t T CO 05 O TT TT u_ V CO -X' CO CO T T C\l r_ TT co « • c j • . u"t • • « c- • co 00 « v w r - tn ~* _o *-* CJ 00 TO CJ tf". ^  T m m C^J C-J g> rw TT in m 0 CJ O ,n C-J 1 1 *-* m *^ cn • i<7*i • . - c o • o iX- CO •'T CO TT o rt ,jj v H g;. o co n C"i T ^ •H CJ tn tr, TT 1*1-1 iV". tf. I 1*1 O T T O CJ O OJ C\J -X" —' 'X' _ i 0. T C'J T T ^ T CJ ~- r--i cr> co _r. r - o:< f-i u*. CJ » • • T . . . T m C-J •>-* cj tn in T U'I CM •*-• u_> •>-' tf". -x u"r en in CJ m TO i-t fw r-- f^ - i^- r— r— r-- r-- rw r— -•- r— \^ r— r— r-- r-— r-- r-- r - r— r— N- r— r-- r— r-.r-~.f--r-. r— r— r - r-- r>- r-- r-~ r-- r-- r - f— f'- r— r— r-- r^ - r-- r-- r~ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 •X1 >X> iX* m m i n r>-i f»- r - r - i - r - N f1- r ' X __> iX> U_> iX" __' -X1 _.' 'X' 'X >X' 'X __• "X1 X ' "X "X1 'X' UT" U_* <X' 'X X ' TO ^ ^> ^ <£> ^ <X> ^ ^ ^ ^ US ^> k£> i£) <X> >X> iX' r>~i r>~i tn f<"i i n in tn tr, m rn m m t>~, in rn m t>~i tn f>*t m m m m rn m m m fr*i m tr, t>-, tn m i n tn tn f>~, tn tn m rn )"'"i in m tn - ~ - ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ _ ^ ^ ^ _ ^ ^ ^ ^ - _ f _ r w ^ ^ ^ ^ ^ ^ ^ ^ ^ . ^ | . ^ ^ ^ ^ r - ^ • ^ ^ r v . ^ ^ w ^ o o 0 0 o o o 0 0 o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o 0 0 o 0 0 o 0 0 o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 c * o o O O O T T O T T C n t f ^ i T ' - ' - ^ ' - ^ - ' - ^ ' - ^ * " - * - ' - ^ LfiLA U % L ^ V T T r o O O O O O O O O T O « > T O T O T O 0 5 T O _ 0 _ 0 _ 5 _ 5 u _ > u 5 * X ' ^ ' O Cg CJ CJ CJ C_ CSJ CJ CJ. CJ C_ CJ CJ C . OJ CsJ eg cj CJ eg c j c j CJ eg CJ CJ CJ CJ C-J CJ CJ CJ CJ CJ CJ CJ eg cj CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ CJ eg CSJ rg CJ C\" CJ CJ CJ CJ CJ CJ eg CJ CJ C-J CJ CJ CJ w 1-1 eg eg CJ eg OJ m m i n T T T T T T T • 0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 0 0 0 '_ „ _ ._ „ „ „ __ _ _ „ O C ' O o c - 0 0 0 0 0 0 o 0 0 0 0 0 0 0 0 0 0 o 0 0 o cr-TO TO X- TO rw r-- r - N- r-- r - r-- co co co co OJ CO TO _ _ _ 0 0 o 0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 0 0 o o o o o o o o o o o o o o o o o o o o o o o eg CJ Csi eg CJ eg eg eg eg e\j CJ eg CJ eg CJ CJ CJ CJ eg eg CJ CJ eg CJ eg eg cj CJ eg cj e tn T Lfi TO r - TO CT< • o OJ m L*I TO r - TO <r- 1 T LTi TO r-— CO #T' tn tn tn tn tr, m tr, rr, fr, tn tn m tr, tn tn tn trt tn tr, in rn m n"i m n-i fr-i n~i (»-i m m tn tr, m m m m tr, tr, tn tn tn tn tn tr, tn tn tn tn tn in m tn R e c o r d A u t h o r A u t h . N o . S p e c i e s S t a g e L e n g t h MM H e i g h t g TeMp. C PatM MMHg D e p t h M 7. H o r t . T i n e h r TGP 0 2 MMHg N2 MMHg pH20 MMHg 365 1 2 . 0 0 8 12 1 2 101 0 11. 1 736 0 . 1 7 30 3 3 . 6 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 3 6 6 1 2 . 0 0 8 12 1 2 101 0 11 . 1 736 0 . 1 7 50 4 6 . 8 114 111 .26 7 1 7 . 1 1 9 . 3 0 3 367 1 2 . 0 0 8 12 1 2 101 0 11 . 1 736 0 . 1 7 - 7 0 6 7 . 2 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 368 1 2 . 0 0 8 12 1 C 101 0 I t . 1 736 0 . 1 7 60 5 5 . 2 114 111 .26 7 1 7 . 1 1 9 . 3 0 9 3 6 8 1 2 . 0 0 9 12 1 2 32 0 11 . 1 736 0 . 1 7 40 7 4 . 4 114 111 .26 7 1 7 . 1 1 9 . 3 0 9 370 1 2 . 0 0 9 12 1 2 82 0 11 . 1 736 0 . 1 7 56 144 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 371 1 2 . 0 0 9 12 1 2 82 0 11 . 1 738 0 . 1 7 25 45 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 372 1 2 . 0 0 9 12 1 2 82 0 11 . 1 736 0 . 1 7 10 2 7 . 6 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 373 1 2 . 0 0 9 12 1 2 82 0 1 1 . 1 736 0 . 1 7 50 1 0 3 . 2 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 374 1 2 . 0 0 9 12 1 2 82 0 11 . 1 736 0 . 1 7 20 3 3 . 4 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 375 1 2 . 0 0 9 12 1 2 32 0 11 . 1 736 0 . 1 7 30 5 1 . 6 114 111 .26 7 1 7 . 1 1 9 . 9 0 9 376 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 100 72 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 377 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 60 3 7 . 2 122 1 1 2 . 7 3 7 7 4 . 4 7 9 . 9 0 9 378 1 2 . 0 1 12 1 2 9 ? 0 11 . 1 736 0 . 1 7 70 3 9 . 6 122 112 .73 7 7 4 . 4 7 9 . 9 0 9 379 1 2 . 0 1 12 i o 97 0 11 . 1 736 0 . 1 7 40 2 8 . 8 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 380 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 25 24 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 381 1 2 . 0 1 12 1 2 97 0 11 . 1 738 0 . 1 7 10 9 . 6 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 382 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 30 2 5 . 2 122 112 .73 7 7 4 . 4 7 9 . 9 0 9 3 8 3 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 80 4 4 . 4 112 .73 7 7 4 . 4 7 9 . 9 0 9 384 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 20 £.£ . O 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 385 12 .01 12 1 2 97 0 11 . 1 736 0 . 1 7 50 3 2 . 4 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 3 8 6 1 2 . 0 1 12 1 2 97 0 11 . 1 736 0 . 1 7 90 5 2 . 8 122 112 .78 7 7 4 . 4 ? 9 . 9 0 9 387 12 .011 12 1 2 38 0 11 . 1 736 0 . 1 7 100 96 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 3 8 8 12 .011 12 1 2 96 0 1 1 . 1 736 0 . 1 7 60 4 5 . 6 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 3 8 9 12 .011 12 1 2 96 0 11 . 1 736 0 . 1 7 80 60 122 112 .73 7 7 4 . 4 7 9 . 9 0 9 390 12 .011 12 1 2 96 0 11 . 1 736 0 . 1 7 30 2 3 . 3 122 112 .78 7 7 4 . 4 7 9 . 9 0 3 391 12 .011 12 1 2 96 . 0 11 . 1 736 0 . 1 7 25 2 6 . 4 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 392 12 .011 12 1 2 96 0 11. 1 736 0 . 1 7 50 3 8 . 4 122 112 .78 7 7 4 . 4 7 9 . 9 0 3 3 9 3 12 .011 12 1 2 96 0 11 . 1 736 0 . 1 7 70 5 2 . 8 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 394 1 2 . 0 1 1 12 1 2 96 0 11 . 1 736 0 . 1 7 40 3 2 . 4 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 395 12 .011 12 1 2 96 0 11 . 1 736 0 . 1 7 10 1 0 . 3 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 396 12 .011 12 1 <L 96 0 11 . 1 736 0 . 1 7 20 24 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 397 1 2 . 0 1 1 12 1 2 96 0 11. 1 736 0 . 1 7 90 6 7 . 2 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 398 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 25 3 4 . 2 122 112.78 7 7 4 . 4 7 9 . 9 0 9 399 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 30 3 7 . 2 122 112 .73 7 7 4 . 4 ? 9 . 9 0 3 400 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 40 4 3 . 2 122 112.78 7 7 4 . 4 ? 9 . 9 0 9 4 0 1 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 70 60 122 112 .73 7 7 4 . 4 7 9 . 9 0 9 402 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 50 5 0 . 4 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 4 0 3 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 10 2 6 . 4 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 4 0 4 12 .012 12 1 C 95 0 11 . 1 736 0 . 1 7 20 3 1 . 2 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 405 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 87 6 3 . 6 122 1 1 2 . 7 8 7 7 4 . 4 7 9 . 9 0 3 406 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 80 66 122 1 1 2 . 7 8 7 7 4 . 4 7 9 . 9 0 9 407 12 .012 12 1 2 95 0 11 . 1 736 0 . 1 7 60 5 5 . 2 122 1 1 2 . 7 8 7 7 4 . 4 7 9 . 9 0 9 408 12 .013 12 1 2 45 0 11 . 1 736 0 . 1 7 64 192 122 1 1 2 . 7 3 7 7 4 . 4 ? 9 . 9 0 9 409 12 .013 12 1 2 45 0 11. 1 736 0 . 1 7 30 90 122 1 1 2 . 7 3 7 7 4 . 4 7 9 . 9 0 9 410 12 .013 12 1 2 45 0 11. 1 736 0 . 1 7 10 5 2 . 8 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 411 12 .013 12 1 <L 45 0 11. 1 736 0 . 1 7 25 7 9 . 8 122 1 1 2 . 7 8 7 7 4 . 4 7 9 . 9 0 3 412 12 .013 12 1 2 45 0 11 . 1 736 0 . 1 7 50 156 122 112 .73 7 7 4 . 4 7 9 . 9 0 9 413 12 .013 12 1 2 45 0 11. 1 736 0 . 1 7 60 130 122 112 .78 7 7 4 . 4 7 9 . 9 0 3 414 12 .013 12 1 2 45 0 11 . 1 736 0 . 1 ? 40 110.4 122 1 1 2 . 7 ? 7 7 4 . 4 7 9 . 9 0 9 415 12 .013 12 1 ^ 45 0 11. 1 736 0 . 1 ? 20 6 9 . 6 122 112 .78 7 7 4 . 4 7 9 . 9 0 9 416 12 .014 12 2 2 77 0 11. 1 736 0 . 6 1 25 2 9 . 4 1 2 4 . 6 1 8 4 . 4 1 7 2 2 . 8 4 9 . 9 0 3 Record Author fluth. No. Species Stage Length MM Height g TeMp. C PatM MMHg Depth M Z Mort. fine hr TGP ;; 02 MMHg N2 MMHg pH20 MMHg 417 12.014 12 2 2 77 0 11. 1 736 0.61 70 54 124.6 184.41 722.34 9.909 4 IS 12.014 12 2 O c 7? 0 11. 1 736 0.61 20 27.6 124.6 134.41 722.84 9.909 419 12.014 12 2 2 77 0 11. 1 736 0.61 40 38.4 124.6 184.41 722.84 9.909 420 12.014 12 2 C 77 0 11. 1 736 0.61 80 57.6 124.6 184.41 722.34 9.309 421 12.014 12 2 2 77 0 11. 1 736 0.81 60 48 124.6 134.41 722.84 9.909 422 12.014 12 2 2 77 0 11. 1 736 0.61 30 31.2 124.6 184.41 722.34 9.909 423 12.014 12 2 2 7? 0 11. 1 736 0.61 50 43.2 122 132.03 694.16 9.909 424 12.014 12 2 2 77 0 11. 1 736 0.61 100 69.6 124.6 184.41 722.34 9.909 425 12.014 12 2 2 77 0 11. 1 736 0.61 90 64.8 124.6 184.41 722.84 9.909 426 12.014 12 2 2 77 0 11. 1 736 0.61 50 43.2 124.6 184.41 722.34 9.909 427 12.014 12 2 77 0 11. 1 736 0.61 10 21.6 124.6 184.41 722.34 9.909 428 12.015 12 4 2 69 0 11. 1 736 0.61 90 69.6 122 178.32 711.37 9.909 429 12.015 12 4 2 69 0 11. 1 736 0.61 10 23.8 122 173.32 711.3? 9.903 430 12.015 12 4 2 69 0 11. 1 736 0.61 50 50.4 122.2 178.32 711.3? 9.309 431 12.015 12 4 2 69 0 11. 1 736 0.61 70 60 122 178.32 711.37 9.909 432 12.015 12 4 2 69 0 11. 1 736 0.61 20 31.2 122 178.32 711.37 9.903 433 12.015 12 A 2 69 0 11. 1 738 0.61 60 55.2 122 178.32 711.37 9.909 434 12.015 12 2 69 0 11. 1 736 0.61 80 64.8 122 178.32 711.37 9.909 435 12.015 12 A ~i c 69 0 11. 1 738 0.61 30 33.4 122 178.32 711.37 9.909 436 12.015 12 2 69 0 11. 1 736 0.61 25 34.8 122 178.32 711.3? 9.903 437 12.015 12 2 69 0 11. 1 736 0.61 40 43.2 122 178.32 711.37 9.909 438 12.016 12 1 2 99 0 11. 1 736 0.61 30 84 122 178.32 711.37 9.909 439 12.016 12 1 2 39 0 11. 1 736 0.61 50 62.4 Itic. - <- 178.32 711.37 9.909 440 12.016 12 1 2 99 0 11. 1 736 0.61 25 42.6 122 178.32 711.37 9.909 441 12.016 12 1 2 99 0 11. 1 736 0.61 30 48 122 178.32 711.37 9.909 442 12.016 12 1 2 99 0 11. 1 736 0.61 90 91.2 122 178.32 711.37 9.909 443 12.016 12 1 2 99 , 0 11. 1 736 0.61 100 120 122 178.32 711.3? 9.909 444 12.016 12 1 c 99 0 11. 1 736 0.61 70 76.8 122 178.32 711.37 9.909 445 12.016 12 1 2 39 0 11. 1 736 0.61 20 37.2 122 178.32 711.37 9.909 446 12.016 12 1 2 99 0 11. 1. 736 0.61 60 69.6 122 178.32 711.37 9.909 44? 12.016 12 1 2 99 0 11. 1 736 0.61 40 55.2 122 178.32 711.37 9.909 448 12.016 12 1 2 99 0 11. 1 736 0.61 50 62.4 122 178.32 711.37 9.909 449 12.016 12 1 2 99 0 11. 1 736 0.61 10 21.6 122 178.32 711.37 9.909 450 12.017 12 1 2 82 0 11. 1 736 0.61 80 34 122 178.32 711.37 9.909 451 12.017 12 1 82 0 11. 1 736 0.61 80 144 122 178.32 711.37 9.909 452 12.017 12 1 2 82 0 11. 1 736 0.61 10 23.8 122 178.32 711.37 9.909 453 12.017 12 1 2 82 0 11. 1 736 0.61 25 41.4 122 178.32 711.37 9.903 454 12.017 12 1 i ' 32 0 11. 1 736 0.61 30 45.6 122 178.32 711.37 9.909 455 12.017 12 1 2 82 0 11. 1 736 0.61 90 153.6 122 178.32 711.37 9.909 456 12.01? 12 1 2 32 0 11. 1 736 0.61 50 69 122 178.32 711.37 9.309 457 12.017 12 1 2 82 0 11. 1 736 0.61 20 37.2 122 178.32 711.37 9.309 458 12.017 12 1 2 82 0 11. 1 736 0.61 50 67.2 122.2 178.32 711.37 9.909 459 12.017 12 1 2 82 0 11. 1 736 0.61 70 103 122 178.32 711.37 9.909 460 12.017 12 1 c 82 0 11. 1 736 0.61 40 54 122 178.32 711.37 9.909 461 12.017 12 1 2 82 0 11. 1 736 0.61 100 168 122 178.32 711.37 9.909 462 12.018 12 1 2 54 0 11. 1 736 0.61 20 56.4 122 178.32 711.37 9.909 463 12.018 12 1 2 54 0 11. 1 736 0.61 70 124.8 122 178.32 711.37 9.309 464 12.018 12 1 2 54 0 11. 1 736 0.61 30 69.6 122 178.32 711.37 9.909 465 12.018 12 1 2 54 0 11. 1 736 0.61 40 80.4 122 178.32 711.37 9.309 466 12.0IS 12 1 2 54 0 11. 1 736 0.61 90 160.3 122 173.32 711.37 9.909 467 12.018 12 1 •y 54 0 11. 1 736 0.61 60 103 122 178.32 711.37 9.909 468 12.018 12 1 2 54 0 11. 1 736 0.61 30 136.3 173.32 711.37 9.909 ord fiuthor Ruth. No. Species Stage Length HM Ueight g TeMp. C PatM MMHg Depth M 7. fort. TiMe hr TGP 7. 02 MMHg N2 MMHg pH20 MMHg 469 12.018 12 1 54 0 11.1 736 0.61 50 93.6 122.2 178.32 711.37 9.909 470 12.018 12 1 2 54 0 11.1 736 0.61 50 94.2 122 178.32 711.37 9.309 471 12.018 12 1 2 54 0 11.1 736 0.61 25 63 122 178.32 711.37 9.903 472 12.018 12 1 2 54 0 11.1 736 0.61 10 43.2 122 178.32 711.37 9.309 473 12.019 12 1 --• 40 0 11.1 736 0.17 32 1608 111.8 112.73 639.89 9.909 474 12.019 12 1 2 40 0 11.1 736 0.17 10 1008 111.8 112.78 699.89 9.909 475 12.019 12 1 2 40 0 11.1 736 0.17 20 1344 111.8 112.78 699.83 9.903 476 12.019 12 1 40 0 11.1 736 0.17 25 1452 111.3 112.78 699.83 9.909 47? 12.019 12 1 2 40 0 11.1 736 0.17 25 1452 111.8 112.78 699.89 9.909 478 12.019 12 1 2 40 0 11.1 736 0.17 30 1560 111.8 112.78 639.89 9.909 479 12.02 12 1 2 55 0 11.1 736 0.17 10 67.2 114 111.26 717.11 9.909 480 12.02 12 1 2 55 0 11.1 736 0.17 20 -14.4 114 111.26 717.11 9.909 481 12.02 12 1 2 55 0 11.1 736 0.17 25 -14.4 114 111.26 717.11 9.909 482 12.021 12 1 41 0 11.1 736 0.17 25 -14.4 114 111.26 717.11 9.909 483 12.021 12 1 2 41 0 11.1 736 0.17 20 -14.4 114 111.26 717.11 9.909 484 12.022 12 1 2 83 0 11.1 736 0.17 10 26.4 122 112.78 774.47 9.909 485 12.022 12 1 2 83 0 11.1 736 0.17 60 62.4 122 112.78 774.47 9.909 486 12.022 12 1 O 83 0 11.1 736 0.1? 70 70.8 122 112.78 774.47 9.909 487 12.022 12 1 2 83 0 11.1 736 0.17 50 55.2 122 112.78 774.47 9.909 488 12.022 12 1 2 83 0 11.1 736 0.1? 80 81.6 122 112.78 774.4? 9.909 489 12.022 12 1 2 33 0 11.1 736 0.17 30 42 122 112.78 774.47 9.909 490 12.022 12 1 2 33 0 11.1 736 0.17 90 93.6 122 112.78 774.4? 9.909 491 12.022 12 1 2 83 0 11.1 736 0.17 40 50.4 122 112.78 774.47 9.909 492 12.022 12 1 2 83 0 11.1 736 0.17 20 33.6 122 112.78 774.47 9.909 493 12.022 12 1 2 33 0 11.1 736 0.17 25 37.3 122 112.78 774.47 9.909 494 12.023 12 1 2 55 0 11.1 736 0.17 60 96 122 112.78 774.47 9.909 495 12.023 12 1 2 55 0 11.1 736 0.17 50 69.6 122 112.78 774.4? 9.909 496 12.023 12 1 2 55 0 11.1 736 0.17 40 55.2 122 112.73 774.47 9.909 497 12.023 12 1 2 55 0 11.1 736 0.17 10 24 122 112.78 774.47 9.309 498 12.023 12 1 2 55 0 11.1 736 0.1? 25 38.4 122 112.78 774.47 9.909 499 12.023 12 1 2 55 0 11.1 736 0.17 76 168 122 112.78 774.47 9.303 500 12.023 12 1 C 55 0 11.1 736 0.17 20 33.6 122 112.78 774.47 9.909 501 12.023 12 1 2 55 0 11.1 736 0.17 30 43.2 122 112.78 774.47 9.909 502 12.023 12 1 2 55 0 11.1 736 0.17 70 115.2 122 112.78 774.47 9.909 503 12.024 12 1 2 4? 0 11.1 736 0.17 25 124.3 122 112.78 774.47 9.909 504 12.024 12 1 2 4? 0 11.1 736 0.17 10 64.3 122 112.78 774.47 9.909 505 12.024 12 1 2 4? 0 11.1 736 0.17 30 153.6 122 112.73 774.47 9.909 506 12.024 12 1 2 47 0 11.1 736 0.1? 20 36 122 112.78 774.47 9.909 507 12.025 12 2 73 0 11.1 736 0.17 25 -79.2 102 132.59 608.11 9.909 508 12.025 12 4 2 73 0 11.1 736 0.17 20 -73.2 102 132.59 608.11 9.909 509 12.026 12 .^ 7? 0 11.1 736 0.17 20 -73.2 105 129.55 631.05 9.909 510 12.026 12 4 2 7? 0 11.1 736 0.17 25 -79.2 105 129.55 631.05 9.909 511 12.027 12 4 2 74 0 11.1 736 0.17 25 -64.8 106 129.55 642.53 9.909 512 12.02? 12 4 2 74 0 11.1 736 0.1? 20 -64.3 106 129.55 642.53 9.303 513 12.028 12 4 2 64 0 11.1 736 0.17 20 264 110.8 117.35 688.42 9.909 514 12.028 12 4 2 64 0 11.1 736 0.17 25 238 110.8 117.35 734.32 9.909 515 12.028 12 4 2 64 0 11.1 736 0.17 10 132 110.8 117.35 683.42 9.303 516 12.029 12 4 27 0 11.1 736 0.17 50 480 111.8 112.78 699.39 9.909 51? 12.029 12 4 2 27 0 11.1 736 0.17 25 180 111.8 112.78 699.83 9.909 518 12.029 12 4 2 2? 0 11.1 736 0.17 20 120 111.8 112.73 639.83 9.309 519 12.029 12 4 2 2? 0 11.1 736 0.1? 70 720 111.8 112.78 699.89 9.909 520 12.03 12 4 2 76 0 11.1 736 0.17 25 39.6 111.3 112.78 699.89 9.909 Record Author Auth. No. Species Stage Length MM Height g TeMp. C PatM MMHg Depth M 7. Hort. TiMe hr TGP Z 02 MMHg N2 MMHg pH20 MMHg 521 12.03 12 4 2 76 0 11.1 736 0.17 70 103.2 111.3 112.78 639.39 9.309 522 12.03 12 4 2 76 0 11.1 736 0.17 50 75.6 111.3 112.78 699.89 9.909 523 12.03 12 4 2 76 0 11.1 736 0.17 20 31.2 111.3 112.78 699.83 9.909 524 12.031 12 4 2 69 0 11.1 738 0.17 20 10.8 122 112.73 774.47 3.309 525 12.031 12 4 2 89 0 11.1 736 0.17 50 28.8 122 112.78 774.47 9.909 52$ 12.031 12 4 2 69 0 11.1 736 0.17 70 45.6 122 112.78 774.4? 9.909 527 12.031 12 4 -. 69 0 11.1 736 0.17 25 13.3 122 112.73 774.47 9.909 528 12.032 12 4 2 73 0 11.1 736 0.17 20 8.4 122 112.73 774.47 9.909 528 12.032 12 4 2 73 0 11.1 736 0.17 50 19.2 122 112.73 774.47 9.909 530 12.032 12 4 2 73 0 11.1 736 0.17 25 10.2 122 112.78 774.47 9.909 531 12.032 12 4 2 73 0 11.1 736 0.17 70 36 122 112.73 774.47 9.903 532 12.033 12 2 2 38 0 11.1 736 0.17 20 -86.4 106 129.55 642.53 9.909 533 12.033 12 2 2 38 0 11.1 738 0.17 25 -86.4 106 123.55 642.53 9.909 534 12.034 12 2 2 84 0 11.1 736 0.1? 25 -67.2 106 129.55 642.53 9.909 535 12.034 12 2 2 84 0 11.1 736 0.17 20 -67.2 106 129.55 642.53 9.909 536 12.035 12 2 2 40 0 11.1 736 0.17 25 396 111.8 112.78 699.89 9.909 537 12.035 12 2 2 40 0 11.1 736 0.17 50 720 111.8 112.73 699.89 9.909 538 12.035 12 2 2 40 0 11.1 736 0.17 20 336 111.8 112.73 699.89 9.309 539 12.036 12 2 2 84 0 11.1 736 0.17 20 57.6 111.8 112.78 699.39 9.909 540 12.036 12 2 2 84 0 11.1 736 0.17 70 96 111.8 112.78 699.89 9.309 541 12.036 12 2 2 84 0 11.1 736 0.1? 25 61.2 111.8 112.78 639.89 9.909 542 12.036 12 2 £. 84 0 11.1 736 0.17 50 79.2 111.8 112.78 699.89 9.909 543 12.037 12 2 2 79 0 11.1 736 0.17 20 24 111.8 112.78 699.39 9.909 544 12.037 12 2 2 79 0 11.1 736 0.17 50 69.6 111.8 112.78 699.89 9.909 545 12.037 12 2 2 79 0 11.1 736 0.1? 25 31.8 111.8 112.78 699.89 9.309 546 12.037 12 2 2 73 0 11.1 736 0.17 70 93.4 111.8 112.78 699.89 9.309 547 12.038 12 2 2 163 0 11.1 736 0.61 25 29.4 125.6 179.34 734.32 9.909 548 12.038 12 2 163 0 11.1 736 0.61 70 55.2 125.6 173.84 734.32 9.303 549 12.038 12 2 2 163 0 11.1 736 0.61 20 26.4 125.6 179.34 734.32 9.909 550 12.038 12 2 2 163 0 11.1 . 736 0.61 50 43.2 125.6 179.84 734.32 9.909 551 12.039 12 4 2 152 0 11.1 736 0.61 20 19.2 125.6 179.34 734.32 9.909 552 12.039 12 4 2 152 0 11.1 736 0.81 70 52.8 125.6 179.34 734.32 9.909 553 12.039 12 4 2 152 0 11.1 736 0.61 25 24 125.6 179.84 734.32 9.909 554 12.039 12 4 2 152 0 11.1 736 0.61 50 40.8 125.6 179.84 734.32 9.909 555 12.04 12 2 2 163 0 11.1 736 0.61 20 16 125.2 182.33 728.58 9.909 556 12.04 12 2 2 163 0 11.1 736 0.61 25 19.8 125.2 182.39 728.58 9.909 557 12.04 12 2 'I C 163 0 11.1 736 0.61 70 52.8 125.2 132.89 728.58 9.909 558 12.04 12 2 2 163 0 11.1 736 0.61 50 36 125.2 132.39 728.53 9.909 559 12.041 12 4 2 204 0 11.1 736 0.61 70 52.8 125.2 182.89 728.58 9.909 560 12.041 12 4 2 204 0 11.1 736 0.61 20 16.8 125.2 182.39 728.58 9.909 561 12.041 12 4 2 204 0 11.1 736 0.61 50 38.4 125.2 182.83 728.58 9.309 562 12.041 12 4 2 204 0 11.1 736 0.61 25 20.4 125.2 182.89 728.58 9.909 563 12.042 12 1 2 114 0 11.1 736 0.81 50 42 125.2 132.39 728.58 9.303 564 12.042 12 1 2 114 0 11.1 736 0.61 20 24 125.2 182.89 728.58 9.309 565 12.042 12 1 2 114 0 11.1 736 0.61 25 27 125.2 182.89 728.53 9.909 566 12.042 12 1 2 114 0 11.1 736 0.61 70 54 125.2 182.39 728.58 9.909 56? 13.001 13 1 2 120 16.2 15 760 0.25 10 19.3 117.2 167.03 703.45 12.78? 568 13.001 13 1 2 120 16.2 15 760 0.25 20 21.05 117.2 167.03 708.45 12.73? 569 13.001 13 1 2 120 16.2 15 760 0.25 25 22 117.2 167.03 708.45 12.737 570 13.001 13 t 2 120 16.2 15 760 0.25 100 cc; 117.2 167.03 708.45 12.787 £ 571 13.001 13 1 c 120 16.2 15 760 0.25 50 26.9 117.2 167.03 708.45 12.737 w 572 13.002 13 4 124 20.6 15 760 0.25 25 23.6 117.2 167.03 708.45 12.73? Record Author fluth. No. Species Stage Length HM Ueight g Tetip. C PatM MMHg Depth M *; Mort. TiMe hr TGP Z 02 MMHg N2 MMHg pH20 MMHg 573 13.002 13 4 2 124 20.6 IS 760 0.25 20 27.7 117.2 167.03 708.45 12.787 574 13.002 13 4 C 124 20.6 15 760 0.25 50 33.3 117.2 167.03 708.45 12.737 575 13.002 13 4 •~\ i. 124 20.6 15 760 0.25 10 26 117.2 167.03 708.45 12.787 576 13.002 13 4 2 124 20.6 15 760 0.25 100 40 117.2 167.03 708.45 12.737 577 13.003 13 4 2 130 19.8 15 760 0.25 10 258 111.4 153.7 678.33 12.787 578 13.003 13 4 2 130 19.8 15 760 0.25 50 486 111.4 153.7 678.33 12.787 579 13.003 13 4 •-> C 130 19.3 15 760 0.25 25 335 111.4 153.7 678.93 12.787 580 13.003 13 4 2 130 19.8 15 760 0.25 100 -84 111.4 153.7 678.93 12.787 581 13.003 13 4 2 130 19.8 15 760 0.25 20 310 111.4 153.7 678.93 12.787 582 13.004 13 1 2 120 13.6 15 760 0.25 25 -34 111.4 153.7 673.93 12.737 583 13.004 13 1 2 120 13.6 15 760 0.25 7 732 111.4 153.7 678.93 12.787 584 13.004 13 1 2 120 13.6 15 760 0.25 20 -84 111.4 153.7 678.93 12.737 585 13.004 13 1 2 120 13.6 15 760 0.25 10 -34 111.4 153.7 678.93 12.787 586 13.005 13 1 2 117 16.8 15 760 0.25 25 11.5 122.3 180.37 737.97 12.737 58? 13.005 13 1 2 117 16.8 15 760 0.25 50 13.6 122.9 180.37 737.97 12.737 588 13.005 13 1 2 117 16.8 15 760 0.25 100 32.1 122.9 130.3? 737.97 12.737 539 13.005 13 1 2 117 16.3 15 760 0.25 20 11.1 122.9 130.37 737.97 12.787 590 13.005 13 1 2 117 16.8 15 760 0.25 10 10.6 122.9 180.3? 737.97 12.73? 591 13.007 13 4 2 130 20 15 760 0.25 25 11.7 122.9 130.37 737.97 12.787 592 13.00? 13 4 2 130 20 15 760 0.25 50 14.2 122.9 180.3? 737.97 12.??? 593 13.007 13 4 2 130 20 15 760 0.25 100 23 122.9 130.3? 737.97 12.787 594 13.00? 13 4 2 130 20 15 760 0.25 10 10.3 122.9 130.37 737.97 12.73? 595 13.007 13 4 2 130 20 15 760 0.25 20 11.2 122.9 130.37 737.97 12.78? 596 13.008 13 4 2 130 20 15 760 0.25 SO 430 112.1 154.36 684.83 12.787 597 13.008 13 4 2 130 20 15 760 0.25 25 -84 110 138.33 684.83 12.78? 593 14.001 14 4 2 79 5.8 10 756.5 0.6 20 320 115.5 0 0 9.208 599 14.001 14 4 2 79 5.8 10 756.5 0.6 50 -51 115.5 0 0 9.208 600 14.001 14 4 2 79 5.8 10 756.5 0.6 50 510 115.5 o 0 9.203 601 14.002 14 4 2 79 5.3 12 756.5 0.6 50 408 115.9 o 0 10.517 602 14.002 14 4 2 ?9 5.8 12 756.5 0.6 50 505 115.9 0 0 10.51? 603 14.002 14 4 2 79 5.3 12 756.5 0.6 20 235 115.9 0 n 10.51? 604 14.002 14 4 2 79 5.8 12 756.5 0.6 20 215 115.9 0 0 10.51? 605 14.003 14 4 O 79 5.8 15 756.5 0.6 20 150 116.5 0 0 12.787 606 14.003 14 4 2 79 5.8 15 756.5 0.6 20 156 116.5 0 0 12.73? 607 14.003 14 4 2 79 5.8 15 756.5 0.6 50 305 116.5 0 0 12.787 608 14.003 14 4 2 79 5.8 15 756.5 0.6 50 268 116.5 0 0 12.787 609 14.004 14 4 2 79 5.3 18 758.5 0.6 20 135 116.7 0 0 15.477 610 14.004 14 4 2 79 5.8 IS 756.5 0.6 50 202 116.7 0 n 15.477 611 14.004 14 4 2 79 5.8 18 756.5 0.6 20 107 116.7 0 0 15.47? 612 14.004 14 4 2 79 5.3 18 758.5 0.6 50 258 116.7 0 0 15.477 613 14.005 14 4 2 102 11.7 9 756.2 0.6 50 462 117.2 0 0 8.608 614 14.005 14 4 2 102 11.7 9 756.2 0.6 20 175 117.2 0 0 8.603 615 14.005 14 4 2 102 11.7 9 756.2 0.6 20 108 117.2 0 0 3.603 616 14.005 14 4 2 102 11.7 9 756.2 0.6 50 L- !—1 117.2 0 0 8.603 617 14.006 14 3 2 105 13.9 9 756.2 0.6 50 515 117.2 0 0 8.609 618 14.006 14 3 ^ c 105 13.9 9 756.2 0.6 20 177 117.2 n n 3.609 619 14.006 14 3 2 105 13.9 9 756.2 0.6 20 165 117.2 o 0 8.609 620 14.006 14 3 2 105 13.9 9 756.2 0.6 50 418 117.2 0 0 8.609 621 14.007 14 4 2 102 11.7 12 756.2 0.6 20 158 117.6 n 0 10.51? 622 14.007 14 4 c 102 11.7 12 756.2 0.6 20 141 117.6 o 0 10.517 623 14.007 14 4 o 102 11.7 12 756.2 0.6 50 252 117.6 0 n 10.51? 624 14.007 14 4 2 102 11.7 12 756.2 0.6 50 242 117.6 0 o 10.51? Record Author Auth. No. Species Stage Length MM Height g TeMp. C PatM MMHg Depth M '/. Hort. Tine hr TGP 'f. 02 MMHg N2 MMHg pH20 MMHQ 625 14.008 14 3 2 105 13.9 12 756.2 0.6 20 214 117.6 0 0 10.517 626 14.003 14 3 2 105 13.9 12 756.2 0.6 50 395 117.6 0 0 "10.51? 627 14.008 14 3 2 105 13.9 12 756.2 0.6 50 525 117.6 0 0 10.51? 62S 14.008 14 3 2 105 13.9 12 756.2 0.6 20 205 117.6 0 0 10.517 629 14.009 14 4 2 102 11.7 15 756.2 0.6 50 118 117.9 0 0 12.78? 630 14.009 14 4 2 102 11.7 15 756.2 0.6 50 193 117.9 0 0 12.787 631 14.009 14 4 2 102 11.7 15 756.2 0.6 20 92 117.9 0 o 12.737 632 14.009 14 4 2 102 11.7 15 756.2 0.6 20 57 117.9 0 o 12.7S7 633 14.01 14 3 2 105 13.9 15 756.2 0.6 50 470 117.9 0 0 12.787 634 14.01 14 3 2 105 13.9 15 756.2 0.6 20 173 117.9 0 0 12.737 635 14.01 14 3 2 105 13.9 15 756.2 0.6 50 490 117.9 0 0 12.787 636 14.01 14 3 _ 105 13.9 15 756.2 0.6 20 154 117.9 0 0 12.78? 637 14.011 14 4 2 102 11.7 18 756.2 0.6 50 52 118.4 0 n 15.477 633 14.011 14 4 •-• C 102 11.7 18 756.2 0.6 20 39 118.4 0 0 15.477 639 14.011 14 4 2 102 11.7 18 756.2 0.6 20 37 118.4 0 0 15.477 640 14.011 14 4 £. 102 11.7 13 756.2 0.6 50 72 113.4 0 0 15.477 641 14.012 14 3 2 105 13.9 18 756.2 0.6 50 453 113.4 0 0 15.477 642 14.012 14 3 2 105 13.9 18 756.2 0.6 20 162 118.4 0 0 15.477 643 14.012 14 3 2 105 13.9 18 756.2 0.6 50 313 113.4 0 0 15.477 644 14.012 14 3 2 105 13.9 IS 756.2 0.6 20 212 118.4 o o 15.477 645 14.013 14 4 2 95 10.4 9 760.2 0.6 50 193 116.5 0 0 8.609 646 14.013 14 4 2 95 10.4 9 760.2 0.6 50 160 116.5 0 0 8.609 647 14.013 14 4 2 95 10.4 9 760.2 0.6 20 12? 116.5 o 0 8.609 648 14.013 14 4 2 95 10.4 9 760.2 0.6 20 101 116.5 0 0 8.609 649 14.014 14 3 2 110 18 9 760.2 0.6 50 287 116.5 0 0 8.609 650 14.014 14 3 2 110 18 9 760.2 0.6 20 158 116.5 0 0 8.609 651 14.014 14 3 2 110 18 9 760.2 0.6 20 116 116.5 0 0 8.609 652 14.014 14 3 2 110 18 9 760.2 0.6 50 456 116.5 0 n 8.609 653 14.015 14 4 95 10.4 12 760.2 0.6 20 122 116.8 0 0 10.51? 654 14.015 14 4 2 95 10.4 12 760.2 0.6 20 88 116.3 0 0 10.51? 655 14.015 14 4 c 95 10.4 12 760.2 0.6 50 133 116.8 0 0 10.517 656 14.015 14 4 o 95 10.4 12 760.2 0.6 50 211 116.3 0 0 10.517 657 14.016 14 3 2 110 IS 12 760.2 0.6 20 122 116.8 0 0 10.517 653 14.016 14 3 2 110 18 12 760.2 0.6 20 195 116.3 0 n 10.51? 659 14.016 14 3 2 110 18 12 760.2 0.6 50 397 116.3 0 0 10.51? 660 14.016 14 3 2 110 18 12 760.2 0.6 50 603 116.8 0 0 10.517 661 14.017 14 4 2 95 10.4 15 760.2 0.6 50 143 117 0 0 12.78? 662 14.017 14 4 95 10.4 15 760.2 0.6 50 178 117 0 o 12.737 663 14.018 14 4 2 95 10.4 15 760.2 0.6 20 93 117 0 0 12.787 664 14.018 14 4 2 95 10.4 15 760.2 0.6 20 87 117 0 0 12.787 665 14.019 14 4 2 95 10.4 18 760.2 0.6 20 54 116.3 0 0 15.477 666 14.019 14 4 2 95 10.4 18 760.2 0.6 50 113 116.8 0 0 15.477 667 14.019 14 4 2 95 10.4 13 760.2 0.6 50 102 116.3 n 0 15.477 668 14.019 14 4 2 95 10.4 18 760.2 0.6 20 55 116.3 0 0 15.47? 669 14.02 14 4 2 113 16.6 9 760.6 0.6 20 35 121.5 0 0 8.609 670 14.02 14 4 2 113 16.6 9 760.6 0.6 50 -4.5 121.5 0 o 8.603 671 14.02 14 4 2 113 16.6 9 760.6 0.6 50 45 121.5 Pi 0 3.609 672 14.02 14 4 2 113 16.6 9 780.6 0.6 20 23 121.5 0 0 8.603 673 14.021 14 4 113 16.6 12 760.6 0.6 20 27 122.2 o 0 10.517 674 14.021 14 4 2 113 16.6 12 760.6 0.6 20 28 122.2 Ij 0 10.517 675 14.021 14 4 2 113 16.6 12 760.6 0.6 50 40 122.2 0 0 10.517 676 14.021 14 4 113 16.6 12 760.6 0.6 50 44 122.2 0 0 10.51? Record Author Auth. Ho. Species Stage Length MM Ueight g TeMp. C PatM MMHg Depth M 2 Hort. TiMe hr TGP V, 02 MMHg N2 MMHg pH20 MMHg 677 14.022 14 4 2 113 16.6 15 760.6 0.6 20 28 122.3 0 0 12.787 678 14.022 14 4 2 113 16.6 15 760.6 0.6 50 40 122.3 0 0 12.787 679 14.022 14 A 113 16.6 15 760.6 0.6 50 43 122.3 0 0 12.737 680 14.022 14 A 2 113 16.6 15 760.6 0.6 20 30 122.3 0 0 12.787 681 14.023 14 3 2 113 17.1 15 760.6 0.6 50 49 122.3 0 o 12.7y7 682 14.023 14 3 2 113 17.1 15 760.6 0.6 20 34 122.3 0 0 12.787 683 14.023 14 3 2 113 17.1 15 760.6 0.6 50 36 122.3 0 0 12.787 684 14.023 14 3 -t 113 17.1 15 760.6 0.6 20 21 122.3 0 o 12.737 685 14.024 14 4 2 162 40.5 8 756.7 0.6 SO 102 120.7 0 0 8.045 686 14.024 14 4 2 162 40.5 3 756.7 0.6 20 70 120.7 0 o 3.045 687 14.025 14 3 2 209 111.5 a 756.7 0.6 50 45 120.7 o 0 8.045 688 14.025 14 3 2 209 111.5 8 756.7 0.6 20 29 120.7 0 0 8.045 689 14.026 14 1 2 206 95.3 8 756.7 0.6 50 82 120.7 0 o 8.045 690 14.027 14 2 2 167 51.6 fi 756.7 0.6 20 152 120.7 o 0 8.045 691 14.027 14 2 2 167 51.6 8 756.7 0.6 50 235 120.7 0 0 8.045 692 14.028 14 4 2 124 19.3 18 763.3 0.6 50 32 121.6 0 0 15.477 693 14.028 14 4 2 124 19.8 18 763.3 0.6 20 24 121.6 o 0 15.477 694 14.029 14 3 2 121 19.2 18 763.3 0.6 50 37 121.6 0 0 15.477 695 14.029 14 3 2 121 19.2 18 763.3 0.6 20 22 121.6 0 0 15.477 696 14.03 14 1 2 126 23.8 IS 763.3 0.6 50 31 121.6 o 0 15.477 697 14.03 14 1 2 126 23.8 18 763.3 0.6 20 22 121.6 0 0 15.477 698 14.031 14 2 2 119 20.5 18 763.3 0.6 50 46 121.6 0 o 15.477 699 14.031 14 2 2 119 20.5 13 763.3 0.6 20 30 121.6 o 0 15.477 700 14.032 14 4 2 162 40.5 12 756.7 0.6 50 84 121.2 0 0 10.517 701 14.032 14 4 2 162 40.5 12 756.7 0.6 20 61 121.2 0 0 10.517 702 14.033 14 3 2 209 111.5 12 756.7 0.6 50 51 121.2 0 0 10.517 703 14.033 14 3 2 209 . 111.5 12 756.7 0.6 20 30 121.2 0 0 10.517 704 14.034 14 1 2 208 95.8 12 756.7 0.6 50 84 121.2 0 o 10.517 705 14.034 14 1 2 206 95.8 12 756.7 0.6 20 55 121.2 o 0 10.517 706 14.035 14 2 2 167 51.6 12 756.7 0.6 20 195 121.2 0 0 10.517 707 14.035 14 2 2 167 51.6 12 756.7 0.6 50 -25 121.2 o o 10.517 708 14.036 14 4 2 162 40.5 16 758.7 0.6 50 35 121.5 0 0 13.634 709 14.036 14 4 2 162 40.5 16 756.7 0.6 20 27 121.5 0 0 13.634 710 14.037 14 209 111.5 16 756.7 0.6 50 63 121.5 0 0 13.634 711 14.037 14 3 *> 209 111.5 16 756.7 0.6 20 37 121.5 0 o 13.634 712 14.038 14 1 2 206 95.8 16 756.7 0.6 20 54 121.5 0 0 13.634 713 14.038 14 1 2 206 95.3 16 758.7 0.6 50 193 121.5 0 0 13.634 714 14.039 14 2 2 167 51.6 16 756.7 0.6 20 160 121.5 o 0 13.634 715 14.039 14 2 2 167 51.6 16 756.7 0.6 50 -25 121.5 0 0 13.634 716 14.04 14 4 2 162 40.5 20 756.7 0.6 50 40 121.6 0 0 17.534 717 14.04 14 4 162 40.5 20 756.7 0.6 20 28 121.6 o 0 17.534 718 14.041 14 3 2 209 111.5 20 756.7 0.6 20 34 121.6 0 0 17.534 719 14.041 14 3 2 209 111.5 20 756.7 0.6 50 57 121.6 0 0 17.534 720 14.042 14 1 2 206 95.3 20 756.7 0.6 20 40 121.6 0 0 17.534 721 14.042 14 1 2 206 95.3 20 756.7 0.6 50 53 121.6 0 0 17.534 722 14.043 14 2 2 167 51.6 20 756.7 0.6 50 270 121.6 0 0 17.534 723 14.043 14 2 2 167 51.6 20 75-6.7 0.6 20 SI 121.6 o 0 17.534 724 14.044 14 4 2 124 19.8 10 756.3 0.6 50 56 121.4 0 0 9.208 725 14.044 14 4 2 124 19.3 10 756.3 0.6 20 31 121.4 n o 9.208 NJ 726 14.045 14 3 2 121 19.2 10 756.3 0.6 20 20 121.4 o o 9.20ft 727 14.045 14 3 2 121 19.2 10 756.3 0.6 50 49 121.4 0 0 9.203 728 14.046 14 1 2 126 23.8 10 756.3 0.6 20 46 121.4 0 n 9.208 Record Author Auth. No. Species Stage Length MM Height g Tenp. C PatM MMHg Depth M 2 Hort. TiHe hr TGF 7. 02 MMHg N2 MMHg pH20 MMHg 729 14.046 14 1 2 126 23.8 10 756.3 0.6 50 90 121.4 n 0 9.203 730 14.047 14 2 2 119 20.5 10 758.3 0.6 50 43 121.4 0 o 9.203 731 14.047 14 2 2 119 20.5 10 756.3 0.6 20 30 121.4 0 0 9.203 732 14.048 14 4 ^* _ 124 19.8 12 756.3 0.6 20 20 122.3 0 0 10.517 733 14.048 14 4 2 124 19.8 12 756.3 0.6 50 33 122.3 o 0 10.517 734 14.049 14 3 2 121 19.2 12 756.3 0.6 20 23 122.3 o 0 10.517 735 14.049 14 3 2 121 19.2 12 756.3 0.6 50 46 122.3 n 0 10.517 736 14.05 14 1 2 126 23.8 12 756.3 0.6 20 31 122.3 0 0 10.517 737 14.05 14 1 2 126 23.8 12 756.3 0.6 50 55 122.3 0 0 10.51? 738 14.051 14 2 2 119 20.5 12 756.3 0.6 20 26 122.3 0 o 10.51? 739 14.052 14 4 2 124 19.8 15 756.3 0.6 20 27 122 0 0 12.787 710 14.052 14 4 2 124 19.8 15 756.3 0.6 50 42 122 0 0 12.737 741 14.053 14 1 2 126 23.8 15 756.3 0.6 20 29 122 0 n 12.737 712 14.053 14 1 2 126 23.8 15 756.3 0.6 50 50 122 n 0 12.787 713 14.054 14 2 2 119 20.5 15 756.3 0.6 50 55 122 0 0 12.787 714 14.054 14 2 2 119 20.5 IS 756.3 0.6 20 31 122 0 0 12.737 745 14.055 14 3 2 121 19.2 15 758.3 0.6 20 41 122 0 0 12.78? 746 14.055 14 3 2 121 19.2 15 756.3 0.6 50 -4.5 122 0 0 12.737 747 14.056 14 4 2 131 26.3 9 755.4 0.6 SO 121 118.3 0 0 8.609 748 14.056 14 4 2 131 26.3 9 755.4 0.6 20 56 118.3 0 0 8.609 749 14.057 14 3 2 124 22.2 9 755.4 0.6 50 226 118.3 0 0 8.609 750 14.057 14 3 2 124 22.2 9 755.4 0.6 20 128 118.3 0 o 8.609 751 14.058 14 1 2 135 32.3 9 755.4 0.6 20 200 118.3 o 0 8.609 752 14.058 14 1 2 135 32.3 9 755.4 0.6 50 440 113.3 0 0 8.609 753 14.059 14 2 2 126 24.1 9 755.4 0.6 20 100 118.3 n 0 8.609 754 14.059 14 2 2 126 24.1 9 755.4 0.6 50 230 118.3 0 0 8.609 755 14.06 14 4 2 131 26.3 12 755.4 0.6 20 73 119.2 0 n 10.517 756 14.06 14 4 2 131 26.3 12 755.4 0.6 50 123 113.2 o 0 10.517 757 14.061 14 3 2 124 22.2 12 755.4 0.6 20 131 119.2 o 0 10.517 758 14.061 14 3 2 124 22.2 12 755.4 0.6 50 250 119.2 0 0 10.517 759 14.062 14 1 2 135 32.3 12 755.4 0.6 20 220 119.2 0 0 10.51? 760 14.062 14 1 2 135 32.3 12 755.4 0.6 50 311 119.2 0 o 10.517 761 14.063 14 2 2 126 24.1 12 755.4 0.6 20 156 119.2 0 0 10.517 762 14.063 14 2 2 126 24.1 12 755.4 0.6 50 276 119.2 n 0 10.51? 763 14.064 14 4 2 131 26.3 15 755.4 0.6 50 96 119.2 0 0 12.73? 764 14.064 14 4 2 131 26.3 15 755.4 0.6 20 76 119.2 0 0 12.787 765 14.065 14 3 2 124 22.2 15 755.4 0.6 20 104 119.2 0 0 12.737 786 14.065 14 3 2 124 22.2 15 755.4 0.6 50 216 119.2 0 0 12.787 767 14.066 14 1 2 135 32.3 IS 755.4 0.6 50 235 119.2 0 0 12.78? 768 14.086 14 1 2 135 32.3 15 755.4 0.6 20 145 119.2 0 0 12.787 769 14.067 14 2 2 128 24.1 IS 755.4 0.6 20 172 119.2 0 0 12.73? 770 14.067 14 2 2 126 24.1 15 755.4 0.6 50 319 119.2 0 0 12.78? 771 14.068 14 4 2 131 26.3 1? 755.4 0.6 20 45 119.6 0 0 15.477 772 14.068 14 4 2 131 26.3 IS 755.4 0.6 50 62 119.6 0 o 15.477 773 14.069 14 3 2 124 22.2 IS 755.4 0.6 20 105 119.6 0 0 15.477 774 14.069 14 3 2 124 22.2 18 755.4 0.6 50 332 119.6 0 0 15.477 775 14.07 14 1 2 135 32.3 13 7S5.4 0.6 50 205 119.6 o 0 15.477 776 14.07 14 1 2 135 32.3 18 755.4 0.6 20 94 119.6 0 0 15.477 777 14.071 14 2 2 126 24.1 IS 755.4 0.6 20 53 119.6 0 0 15.477 15.477 4_ 778 14.071 14 2 2 126 24.1 18 755.4 0.6 50 136 119.6 0 n 779 14.072 14 4 -. 150 32 12 758.4 0.6 50 5 131.4 201.56 781.76 10.517 >j 730 14.073 14 4 2 150 32 12 758.4 0.6 50 4.5 131.4 157.61 325.49 10.517 248 f'_ r- rw rw r- r- f- r- r- r- r- r- r-_ r-_ r- r- r- rw r- r- r~ r- r- r-- r- r- rw r_ r_ rw rw r- rw r- r- co co n- r- r- r- r- r- r- rw r_ r- cr< en rw y. •_-.w--.--.--_.---w f_ r— r— •->:• r— r— _• •___• o —- rr LA b"i LA LA LA b". LA LA LA LA LA LA LA LA LA LA LA LA LA U1 U1 b"i LA LA LA LA LA LA LA LA LA LA LA .w r- Cj fsj tA LA rw r- T T .w T T TO TO- TO b"l © O © © c" © © © O © O O © O © O © © O O O O © © O CD © O © © © © © © eg eg 0^  Cn © © C-J OJ U-l LA b"i 0. LA LA TO CO CO © CO LA tv-r en TO 05 TO *T T tfl TO LA U"i C' 'NJ LA f-J lA fn —« Cn Tw CC' TO LA r- U"i 0"> Tw 0? b"i TO m 0? O O O O O O O O O O © O O © O O O © © *T TO >T> r-i LA Tw © © 0i © in T rw 'T> r- TO TO tn T CJ LA n- m TO rn • TO © T CO TO LA in -T or' rw ©eg ^ ^  TO --. cn u*" <r" LA rr TO © TT CJ TO -n rw rw TO <A r- u-i TO CJ in *r T TO TO TO rw C-J •—< —' LA b". LA TO 0:> rj> 7' M V fr( TT o TO CT. TO © © *»-• T CO LA CJ TO CO TO CJ rw r_ rw CC' cn rw rw r_ rw rw rw rw rw co U"i co o:< o:- cr- cn u" r- co o:> co co cr. rw -w TO rw rw • —» TO © © O © © © © © O © © © © i o. LA eg m rr T TO TO LTI O LA cn rn *-i TO in LA CO r- rn eg eg TO ej cn m cr. m in cn I © LA r- © 0". -T' CO i-i TH LA •T TO OJ OJ -~H TO- LA TO CJ LA •«-* © T- TO TO- TO •-* rw cn CJ c-j *—> CJ -»-• i-t -P-I — eg CJ — w T CJ eg O-J eg TH eg OJ m eg tn ' TO' TO- TO- TO —< *-» T-> • , U"i CT' 1 LA Le. LA LA TO • CO O'.i • —t TO i b-< u-i CJ rw ti-, o-i f,-, ,n CJ eg CJ eg eg CJ eg eg eg T T T -r TT rr rn t>- m in m I---I »n en in in T T T ri^H--iH-Hri--i-^ - - . -—• eg eg eg cj eg > LTi eg eg r- u-. o eg r>- LA LA O-J en tw LA eg eg rw en r . . . . . CJ t-H • . p . . . . . LA "T T in TO TO TO' CO LA T f J ^ w m H c. in TO Lfi u-i LA Lfi r- cn eg © •»-' ©' in eg Lfi TT ' - • • • • • > rw eg (n m m in in in • eg «H eg ui TT - - eg rn i t i i i i • TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO1 TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO <Z- O <Z' CZ- CL' ci ^ CZ' c£ © © © © © © ' © © ©' O O O O O O O O ^ CZ' ci> & CZ> <Z- CZ' c£ © TO O O O O © -.3 O O CO O? CO CC U"i LA U*i Lf Tw fw fw fw CO TO CO CO 0_> CO CO CO CO CO 00 CO TO CO CO CO CO TO CO CO CO TO 0? CO CO 05 TO TO 05 © © TO TO' _ LA LA LA Lfi LA Lfl U*t b"i LA LA bl Lfi b'i LA U"i Lfi U"i LTi LA LA Lfi Lfi b"i Lfl LA Lfl Lfi U't LA TO TO b"i Lfi LA . rw rw rw rw r- fw rw rw rw r- rw . r  rw rw rw rw rw r- rw . r_ rw r-- r- rw rw r_. rw rw TO TO TO TO TO © © © © © © © TO U"i LA U*i UTt LA U-i TO TO' TO' TO' TO TO TO Lf. fw rw fw rw F_ r_ rw r_ N M - N rw fw eg cj CJ eg eg -rg eg eg CJ eg eg cj eg eg eg eg eg eg eg eg eg CJ OJ CJ CJ eg CJ eg OJ CJ OJ eg CJ LA U"I © © eg eg LA LA CO CO CO U-I OO CO en cn v eg co e^J eg eg CJ eg eg eg eg eg eg eg eg eg eg eg eg eg CJ OJ CJ eg eg eg CJ OJ OJ O-J OJ OJ eg CJ OJ CJ CO CO UI LA u_t LA LA U*I LA LA b*. Lfi Lfi b'i IA U-I b-. co rn rn in rn in rn rn rn in rn rn in t<~\ r-i m r-_i in m r>" in m in m in in r>- in rr-( rn rn ^ r»-i rn « Hrtrt-HHH-^rt-H-H-HrtH-H^r.U'i r*+-4vM*-4v-lT-iw**~i^* W f-l w-l O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O C n c n c n ^ C . 0". mcr. cn (T> ff- <T> <T. <T' CHTO Li"i Lfi U"l L_"r Lfi Lfl IXi Lf*i LA LA LA LA Lfi U'l b^  L_Ti y eg eg CJ eg eg eg eg CJ eg eg eg CJ eg eg CJ CJ CJ CJ eg eg eg eg eg eg eg eg eg eg eg eg eg OJ eg eg eg eg eg eg OJ OJ eg eg eg eg eg eg CJ CJ eg eg cj eg • ^ • ^ t r r - - - - r r T T T T T T T - T * > T T T Ti/iTOrwTOiT^«^cg^iT_ATOr-cocr. O-I-»-H w f ^ T W y ^ ^ 0 - P ^ r t c g l r 1 T l J ' g . ^ ^ Q a l f f ' < 3 ^ H H H H H ^ J r l n 7 , T T r u H \ l rw F- rw rw rw rw © OJ TO TO co co co co o:- © en cr. <n cn cr. c*. cn <r< cn • © © © © © © © © © © © © - - ' • - * • - « • - < - - ' — . - - . - H - ^ — W W . © © © © © © . © © © © © © © © C> . © © © © C ' © © O C ' ^ ' ^ ^ ^ ^ - ^ ^ ' ^ - ^ ^ ^ ^ ' — • ••rH'— <r* —* rH -H ^  -H-— ^  — eg tn T u. h CO CO CO CO 03 oo o r_ p- rw -w rw rw r<-TO cr- © — eg rr-i ••r b"i TO rw co a- © '-t eg r>" T Lft TO rw TO cn © eg rn T Lfi TO rw o:- cn © eg rn T U'I TO rw o:- c-- o *-< CJ ' oo oo cn cn c> &• IT- cn cr» cn CT< cn © © © © © © © © © © i-i *-—• *•-* * •—« *^  eg cj OJ CJ eg oj c-j eg OJ OJ in in ^  r_ rw rw r- rw N. rw rw rw rw r- rw co co co TO TO co co co co TO TO co TO OZ< TO CO TO TO TO co co » co co co co co co TO CO TO TO CO 249 LTi LTI LTI ' X ' -X- >X> - X ' «X» tX> >X' ' X - <X- ' X ' <X> >X' <X> X> < X <X- ' X 1 a " ' j J u J ^ ' -X- u ) a> X> ^ ^ ' j ? - i ' a"> ' i ) U > U > a " ' a - - u ? u." u 3 X ' X - CM CM CM CM CM CM a - c o c o c o r - r*- r - r - r - r - r - r*- x - x> ^x- x - <x> •x- <x- r- iX> r - r - rw t— r - r-- r%- r - r - r - r— r_ r - r - u*i L n LA u i ix> r - <x> ^ ^ ^ rt f \ j 0 0 <\j f s j V-M CM CM rtO-Jrtrt*-<»-i»-irtrtTHrt—<»-irtrtrtrt--H m fn *•« * H CM CM m LTi U ' I LTI LTI CM CM C-J CM U ' l l / i U ' l l / i T T 7 T 7 CM CM m CM ft"! P - r - X > «X' ' X - «X< " X ' >X> T V ^ *T ^ T T e n 0 " . CJj O - . £r. V m H H H ^ x> f£ !J38¥ }£ g g $ - £ ^  g £j K £ S | - 2 1 TO ^ 1 j£ 1 - S£ * i j ^ u * . ^ r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t * ~ l " ^ ^ , ^ •**!! j 'r'j l*^ > '*^' ""^ ""^ ^ * * ^ **^' **^> ^ * ""^ *^*' **^' *^*> ""^ ' " ^ **^ "' **^ "' **^ "' '**'' ' " ^ '7^ '~1 pj ' ~ ~ d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d * = ; ~ ' H ~ ~ ' ~ ^ ^ U ^ O O O O O O O O O O O O O C ' O C ' O O O O O C ' O O O O O O O O O O O O O O O O O O C - O O r n O O C - O ' a s S S S S S S S S S S S S S S S S S S S S f f i S 8 S « S 8 8 8 B ! S f f i S « * | § | | S S S S S S Cr. o O O O O Od CM C \ l CM CM CM CM CM CvJ C\J CM CM C ^ Record Author Auth. No. Species Stage Length HM Height g TeMp. C PatH MMHg Depth M Z Hort. TiMe hr TGP '/. 02 MMHg N2 MMHg pH20 MMHg 93? 22.002 22 2 2 160 51 12 754 0.6 7 643 116.8 0 0 10.517 933 22.003 22 4 2 200 74 12 754 0.6 50 456 116.3 0 0 10.51? 939 22.004 22 4 2 200 74 12 754 0.6 50 43 121.2 0 0 10.517 940 22.005 22 3 2 140 36 12 754 0.6 50 38 121.2 0 n 10.517 941 22.006 3 2 140 36 12 754 0.6 50 360 116.8 0 0 10.517 942 22.007 22 3 2 160 51 12 754 0.6 50 -64.8 111 0 0 10.517 943 22.003 22 3 2 140 36 12 754 0.6 50 -64.8 111 0 0 10.51? 944 22.009 22 4 C 200 74 12 754 0.6 50 -64.8 111 o 0 10.517 945 22.01 22 2 2 160 51 12 754 0.6 20 -64.8 111 0 0 10.517 946 22.011 22 2 2 160 51 12 754 0.6 20 -64.8 116.8 0 o 10.517 94? 22.012 22 2 2 160 51 12 754 0.6 20 50 121.2 o o 10.517 948 22.013 22 2 2 160 51 12 754 0.6 25 -64.8 111 0 0 10.517 949 22.014 22 <•> 2 160 51 12 754 0.6 25 -64.8 116.8 o n 10.51? 950 22.015 22 2 2 160 51 12 754 0.6 25 60 121.2 0 o 10.517 951 22.016 22 3 2 140 36 12 754 0.6 20 -64.8 111 0 0 10.517 952 22.017 22 3 2 140 36 12 754 0.6 20 195 116.8 0 0 10.517 953 22.018 22 3 2 140 36 12 754 0.6 20 25 121.2 o 0 10.517 954 22.019 22 3 2 140 36 12 754 0.6 25 -64.8 111 0 0 10.517 955 22.02 22 3 2 140 36 12 754 0.6 25 230 116.8 o 0 10.51? 956 22.021 22 3 2 140 36 12 754 0.6 25 26 121.2 0 0 10.517 95? 22.022 22 4 2 200 74 12 754 0.6 20 -64.8 111 0 0 10.517 958 22.023 22 4 2 200 74 12 754 0.6 20 210 116.8 0 o 10.51? 953 22.024 22 4 2 200 74 12 754 0.6 20 35 121.2 0 0 10.517 960 22.025 22 4 2 200 74 12 754 0.6 25 -64.8 111 0 0 10.517 961 22.026 22 4 2 200 74 12 754 0.6 25 230 116.8 0 0 10.517 962 22.027 22 4 2 200 74 12 754 0.6 25 36 121.2 0 0 10.517 963 23.001 23 3 2 193 . 95.5 12.5 754 0.28 80 504 113.4 0 0 10.869 964 23.002 23 3 2 193 95.5 12.5 754 0.28 60 504 118.4 n 0 10.869 965 23.003 23 3 2 193 95.5 12.5 754 0.23 10 504 116.4 0 o 10.863 966 23.004 23 3 2 193 95.5 12.5 754 0.28 50 -50.4 116.4 0 0 10.869 96? 23.005 3 2 193 95.5 12.5 754 0.28 20 -50.4 116.4 n 0 10.363 968 23.006 23 3 2 193 95.5 12.5 754 0.28 50 -50.4 114.4 0 0 10.869 969 23.00? 23 3 2 193 95.5 12.5 754 0.28 20 -50.4 114.4 0 0 10.869 9?0 23.008 23 3 2 193 95.5 12.5 754 0.28 50 -50.4 111.4 0 0 10.869 971 23.009 23 3 2 193 95.5 12.5 754 0.28 20 -50.4 111.4 o 0 10.869 972 23.01 23 3 2 193 95.5 12.5 754 0.23 50 -50.4 109.4 0 0 10.869 973 23.011 23 3 2 193 95.5 12.5 754 0.23 20 -50.4 109.4 o 0 10.369 974 24.001 24 5 3 235 121.5 12 760 0.6 50 11 131.4 0 o 10.517 975 24.002 24 5 3 235 121.5 12 760 0.6 50 20 127.4 0 0 10.517 976 24.003 24 5 3 235 121.5 12 760 0.6 50 29 125.4 0 0 10.517 977 24.004 24 cr 3 235 121.5 12 760 0.6 50 97 121.4 0 0 10.517 978 24.005 24 c 3 235 121.5 12 760 0.6 50 42 122.4 0 0 10.51? 979 24.006 24 5 3 235 121.5 12 760 0.6 50 265 119.4 0 0 10.517 980 24.007 24 c. 3 235 121.5 12 760 0.6 50 -50.4 115.4 0 0 10.517 981 24.008 24 5 3 235 121.5 12 760 0.6 50 -50.4 113.4 0 0 10.517 982 24.009 24 5 3 235 121.5 12 760 0.6 50 67 121.4 0 0 10.51? 933 24.01 24 q 3 235 121.5 12 760 0.6 50 172 119.4 0 0 10.517 984 24.011 24 c 3 235 121.5 12 760 0.6 50 -50.4 116.4 0 0 10.51? 935 24.012 24 5 3 235 121.5 12 760 0.6 50 -50.4 113.4 o 0 10.517 936 24.013 24 c; 2 67 Ti.5 12 760 0.6 50 97 122.4 o 0 10.517 987 24.014 24 5 2 67 3.5 12 760 0.6 50 170 119.4 0 0 10.51? 933 24.015 24 5 z 6? 3.5 12 760 0.6 50 349 115.4 0 0 10.517 251 co © co co co co co co co co co « co » w cc o:> © » w co co o:> 05 a- « » T v T T T T T r- ^ T ^ r- r- T m r- rw rw rw rw r- r- r~ r-o >=• cz- o o o C' o o o o o cr- <=:• <=:• •=>>_:• cz- cr> cz- ro ro ro ro ro ro co o:- co fi co co -:o - o co —• —< —.—..-«—._ —. CM '."si CM CM CM -M CM CM <S1 <>1 CM CM CNJ N 'N OJ N M CM CM CM CM CM CM CM >X> -X' >X> © <X' <X< >X> rw N- r— © rw N- rw <X> LA U'l U'l LA U'i LA U'i U'i LA cr" cr* © © © © © © © © © © ©. © cr. © cr- © © © © O*" © O"- © © tA in (r, f'i in (A rn CM CM CM in" CM W W r o W O O O G O O O G C* • O O O O O O © © O ' . o O © O O © © ' m H T m OJ W H H H ^ o i f l O CO G r— CM LTi * H O I-» © r- X> <X< CM CM <-< r- V © r - rw r - rw fw r-- .x> rw r_ rw © rw rw rw ' o © © © © '© © © © o © © © . rt 00 U3 CT- W CO TT U'i CM CO iX> TO rw cc- co © cr- r- x - «• r- en rti rt rt CM CM *-« *-t iM rt rt 1 m © © u 'i m t r u'c u - :• u _i u'i K£< © CM © u'i CM u i t r u'i x> © u'i © © co «x- co -x-CM • iv. • m C M m CM CM • » C O © CT' © CT 1 © rt © rt rt .x> O "X1 © © V ,™i rt (j-. *-i rt rt rt rt rt rt m m (ir, CM m C M i n Ci T fi M W t<~, <>4 C-i C^i C M C-J C M r>**i fn <n ^ U J T O'I C O C M C O M —^* »-t rt M C M C M © © CM CM CM CM © CC' CC © CM LTi r - U'I U'I U'I rw cr. T U"i iX' u!» LA LA U'l U'i U'l U1 LTi LTi P"t U'i U'l *7 -J? rw CM CM f"i ' © in rn t r © v f n LA* r -rt rt rt rt j?, «X> u i rw T o o:> CM © #M tn m r - m m u". rw © co .•..1 . u"i —• • • • • • •in • • • © - - - M Cn rw <p CM fV| —* -X- CM fV| C O rt rt i_n 0-i CM * - * 1. . . . . . . . ^ ^ - ^ U ' . U*I PW U " i U'I C M C M t r ; rt rt rt rt u'i U'i U'i LTi LTI U'I U'I LTI LH U'I LTI ! o o c" © c" o © © © o © © o © 1 T H T H >XI IX» <X> 1X1 1X1 *JL> " X 1 1X1 X ' X ' <X' *X* *X> s£> 1X1 "X* «X' ' X 1 siJ •x* o © c" © © © © c" o © © © © © ©" © © © © © > <X< iX' 'X' X1 <x< o o © © © O O O O O O O O O O O O O O O O O O O O O O O O O O T T T T T ^ ' T T V T T V T T T t r ^ T ' T T T ' T ' T T T r ' T >X> X' 'X> -X' UT» 'X- -X' *X> U 3 *X- X' UD u:- <X- X> s£- "X- X> «X' iX> >X> iX> iX> K£> "X« LTI LTi U'l U'l U'l U'l LTl LCi LTt U'l LTi LTi LTi LTi U'l LTi LH U'i U'i Lfl U'. U1 U'l U't U'l LTi o o © © . o © © © © © © © © © © ' © © © © O © © © © © J> -X' %£• -X- -X) "X- U'l U'i U'i "X> U'i U"i U1 tX» © C M CM C M C M C M C M CM CM . o © o © • © © © © © © © © © © © © © © • - o c> o o o o o rw rw rw fs- r - r - rw rw rw rw rw r - rw rw rw 1 r J rw r - rw r- rw r - fw > © o © © o © o o © © © © © © ©> © U'i fw 03 cr. -X' fw rw u'i in O O O O O O G G O O O O O O O O O O O O O O O O O O O t f t f J ^ ^ r f J > t f J > J ' ^ u 5 ^ u 5 u 5 0 0 0 0 G G O O O O cr. «> co 00 © co co co co © co cc' co co co co i n T t r C M C M © t r cn u:-rt |Xi U1 LTl LCi LH U'i LTl U1 «-< CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM 00 CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM 0-J CM CM CM CM CM CM f i f i n m m i n i n CM t r t r T r t r t r t r t r t r ^ t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r t r f t r n r n r ^ i n m i 1 --! i n m CM rw r_ r - rw fw r - T — rw fw . r«_ rw r_ r- rw . rw r - rw rw rw rw rw r - rw « cr. cn © a> © © © © cr. cr- © CM C M O J C M C M CM CM O J r n r n t r I A ^ i rw rw fw rw rw rw rw rw r - co oooo©.r .oooo©o©©©©©©©©©©©©©'^©o©o©©©o© © © o © © © © © © © © © © © © © © © © © o © © © © © © © © © © © © © © © • © © © © © © © o © c> © o o © © u*i -X' rw co © © ^ C M m t r LTI us rw © © o rt csj r^i t r rw © © o rt CM r n ^ LTi u:- rw © o CM r n t r LTI x> rw co cn o ^ CM r o t r Ln x> ©«©©03©©©©©©©©©©©©©oo©©©©©^ w ^^^^^ w ^ w cMCMCMCMCMCMCMCNj CM C M m t n i n m i n i n r- i « • « © © C O © © C3 » © © © co co © © © © © © © © © • © © © © © © © © © © © © © > © © © © ' © © © © © © © © © © © © R e c o r d A u t h o r A u t h . N o . S p e c i e s S t a g e L e n g t h MM H e i g h t g Tewp. C P a t M MMHg D e p t h M 'i N o r t . T i M e h r TGP 't. 0 2 MMHg N2 MMHg pH20 MMHQ *_39 2 4 . 0 1 6 24 5 2 67 3 . 5 12 760 0 . 6 50 - 5 0 . 4 113 .4 n 0 10.517 390 2 4 . 0 1 7 24 c; 67 12 760 0 . 6 50 54 121 .4 0 n 10.517 991 2 4 . 0 1 8 24 c; 2 67 3 . 5 12 760 0 . 6 50 119 119 .4 0 0 10.517 992 2 4 . 0 1 9 24 5 C 6 ? 3 . 5 12 760 0 . 6 50 315 115 .4 0 0 10.517 993 2 4 . 0 2 24 c; 2 67 3 . 6 12 760 0 . 6 50 - S O . 4 113 .4 0 0 1 0 . 5 1 ? 994 2 4 . 0 2 1 24 5 3 235 1 2 1 . 5 12 760 0 . 6 20 3 131 .4 0 0 10.517 395 2 4 . 0 2 2 24 5 3 235 1 2 1 . 5 12 760 0 . 6 20 16 1 2 7 . 4 0 0 10.517 996 2 4 . 0 2 3 24 c; 3 235 1 2 1 . 5 12 760 0 . 6 20 20 1 2 5 . 4 0 0 10.517 997 2 4 . 0 2 4 24 5 3 235 1 2 1 . 5 12 760 0 . 6 2 0 64 121 .4 0 0 10.517 998 2 4 . 0 2 5 24 5 3 235 121 .5 12 760 0 . 6 20 34 1 2 2 . 4 0 n 10.517 999 2 4 . 0 2 6 24 5 3 235 1 2 1 . 5 12 760 0 . 6 2 0 142 1 1 3 . 4 0 0 10 .51? 1000 2 4 . 0 2 7 24 5 3 2 3 5 1 2 1 . 5 12 760 0 . 6 2 0 - 5 0 . 4 1 1 5 . 4 0 0 10.517 1001 2 4 . 0 2 8 24 5 3 235 121 .5 12 760 0 . 6 2 0 - S O . 4 1 1 3 . 4 0 n 10 .517 1002 2 4 . 0 2 9 24 5 3 235 1 2 1 . 5 12 760 0 . 6 2 0 44 1 2 1 . 4 0 0 10.517 1003 2 4 . 0 3 24 5 3 235 1 2 1 . 5 12 760 0 . 6 2 0 1S1 1 1 9 . 4 0 0 10 .51? 1004 2 4 . 0 3 1 24 5 3 235 1 2 1 . 5 12 760 0 . 6 2 0 - 5 0 . 4 1 1 6 . 4 0 0 10.517 1005 2 4 . 0 3 2 24 5 3 235 121 .5 12 760 0 . 6 2 0 - 5 0 . 4 1 1 3 . 4 0 0 1 0 . 5 1 ? 1006 2 4 . 0 3 3 24 5 2 67 3 . 5 12 760 0 . 6 2 0 50 1 2 2 . 4 0 0 10.517 1007 2 4 . 0 3 4 24 5 2 67 3 . 5 12 760 0 . 6 2 0 109 119 .4 0 0 10 .51? 1008 2 4 . 0 3 5 24 5 2 67 3 . 5 12 760 0 . 6 2 0 246 1 1 5 . 4 0 o 1 0 . 5 1 ? 1009 2 4 . 0 3 6 24 5 2 67 3 . 5 12 760 0 . 6 2 0 - 5 0 . 4 113 .4 0 0 1 0 . 5 1 ? 1010 2 4 . 0 3 7 24 5 2 67 3 . 5 12 760 0 . 6 2 0 20 1 2 1 . 4 0 0 10.517 1011 2 4 . 0 3 3 24 5 2 67 3 . 5 12 760 0 . 6 2 0 79 1 1 9 . 4 0 0 10.517 1012 2 4 . 0 3 9 24 5 2 6 7 3 . 5 12 760 0 . 6 2 0 135 115 .4 0 0 10.517 1013 2 4 . 0 4 24 5 *t. C 67 3 . 5 12 760 0.6 2 0 - 5 0 . 4 113 .4 0 0 10.517 to 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0097966/manifest

Comment

Related Items