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I I}: - fyfifl H'b‘xl I ' $.‘y‘1XN‘A:.I‘\?‘I‘o nonnauoa no Hooanz souuoxam :Owumunummuoeam coaumuaumouoonm moan mo.uoma:z .oowunaou o>wumxwm mo annuOHOQEOO one wawhum> .mosowsmoou sowumxww now .Auom~ M.monhv UO- um seams nauseoumouOQOm mom donned advance 0» hum unouu Boosamu anamooxo he vooamuoo sumo Hooamhmm "ounuouooaoy one sowuoxwml uooaquooxm .N manna 36 ~\N as on m m a N: 8 an m m m :~ S 8 m m N Oxm m on m m a oouuoxfim no seam A.:«av A.ofimu . Houuoou Housmafiuomxm onouo Hmuooaauooxo oouoommoss seam mafia ousmooxo roam we Honesz uo>o veaoauoa.mo nooasz sowumxuh :Oaumunuoouooom I. .Aoomq acquOHOo u.swoom OH souumxwm he ooBOHHOw Roma a.munav comm no poems ooumusumouoonm new Hoauoa huounom Ou hum unouu someway meamooxo ho nooumuoo some “moauhmm “mafia soaumxam I N usuaauooxm .m manna 37 The lesions produced in this experiment are considered below, section III, mortality data. C. The Effects of Fixation Techniques on Gas Bubble Size 0n the basis Of these preliminary laboratory investigations, the rapid fixation technique was found useful in laboratory diagnosis Of GBD, but raised questions concerning induction Of artifactual lesions in treated and control fish. Since it is possible that histological appearance Of emphysema may be influenced by fixative solution and temperature and gas pressure, a theoretical examination was also de- velOped to evaluate this phenomenon. Considering that the atmospheric pressure is constant when the fish is transferred from supersaturated water at ambient temperature (12°C) to Bouin's solution at varying temperatures, Charles' Law is applicable: "The volume Of a gas at constant pressure increases proportionately to the absolute temperature." The law may be expressed by the equation Vl/VZ - T1/T2 , where V1 and T1 represent volume of gas bubble and absolute temperature in one case (water) and V2 and T2 the quantities for the same mass Of gas in another (Bouin's solution). For example, if a fish with a 1 cm2 gas bubble were transferred from supersaturated water at 12°C to Bouin's solution at 48°C, the size of the bubble would increase to 1.126 cm , or approxi- mately 12.62 (Table 4). It was determined that the small alteration induced could-be better tolerated:than complete loss of lesions resulting from standard fixation techniques. II. Chronic Gas Bubble Disease: Experiment #3 A. Introduction The high supersaturation level experimental group (Treatment 1) 38 Table 4. Theoretical influence of fixation temperature on GBD lesions: the effect Of varying ambient temperature, 12°C, at constant pressure, on the volume Of a gas bubble during fixation in Bouin's solution. Temperature Bubble size Oc OK cm3 12 285 1 -200 73 .26 -150 123 .46 -100 173 .61 - 50 223 .78 - 20 253 .89 - 10 263 ' .92 - 5 268 ‘ .94 0 273 .96 5 278 ' .98 10 283 .99 20 293 1.03 50 323 1.13 100 373 1.31 39 Of fish experienced a high morbidity and mortality. At the end of the 60-day test, 802 Of the two hundred rainbow trout fry were dead. Signs consistent with GBD were found in nearly all moribund or recently dead fish. Groups Of fish held in Treatment 2, 3 and Control experi- enced no mortality due to GBD or any other disease. Sporadic incidence tissue pathology occurred as frequently in controls. The well water supply at the Aquaculture Laboratory was found to contain supersaturation Of nitrogen. Therefore, the control group levels Of nitrogen ranged around 1052 throughout the test. The total dissolved gas saturation and oxygen saturation in the control trays were below 1002. B. Water Flow Adjustment As a result of utilization Of an initial constant waterflow (200 ml/min), which did not take into account the rapid growth Of the fry, the absolute level Of oxygen in the water inadvertently decreased in proportion to the increased demands of growing fish. This resulted in slightly decreased oxygen tension amounting to five mg/l in the lower (Treatment 3) and control trays. In the remaining trays, there was less Of a decrease. Since the system was not provided with a controlled external oxygen supply tO keep a desirable minimum level (6 mg/l), it was decided tO simply increase the waterflow from 200 to 400 ml/min, on the 46th day Of the 60-day experiment. With the water- flow change, the nitrogen levels stayed approximately the same as before, but oxygen levels increased substantially in both the treatment and control trays. 40 III. Mbrtality Data A. Acute Gas Bubble Disease External signs develOped within a few minutes after the fish had been exposed to the supersaturated water. Gas bubbles adhered to the surface Of the fish body and fish tended to stay at the maximum avail- able depth of the chamber. Shortly thereafter, in approximately 10 minutes, the fish exhibited abnormal behavior including erratic swimming and jumping free Of the water. This was followed by spasmodic spiraling and drilling movements, and swimming side and belly-up. These symptoms culminated in convulsions, the moribund stage, frequently leading to death in approximately 20 minutes. The most common external signs recorded from the gross examination were gas bubbles in the caudal fins. Gross examination Of internal organs was not conducted before fixation. Individual resistance to development of GBD occurred within groups (Tables 2 and 3). In each group Of three experimental fish, usually one fish did not show behavioral changes, and did not die after three hours exposure even though visible gas bubbles were adhered tO the surface Of the body. B. Chronic Gas Bubble Disease 1. Gas Saturation and Lethality Rainbow trout fry were exposed to supersaturated water for 60 days at different experimental gas saturation levels. Plots of proportional mortality against gas saturation for each test treatment and control are shown in Figures 1 through 4. 41 Figure 1. Cumulative mortality curvea for rainbow trout fry (n - 183) exposed to approximately 115 percent total dissolved gas saturation during 60 test-days (Treatment 1). aMortality occurred after two days Of exposure to supersaturated water, reaching 6% in the first 10 days. There was insignificant mortality during the period from.day 10 to day 46. On day 46 the waterflow was changed from 200 to 400 ml/min. Death occurred rapidly at that point and continued, reaching 80% at the termination of the experiment. bNitrogen saturation levels were constant at 117% throughout the experi- ment. Oxygen saturation levels which were approximately 103% at the beginning, diminished gradually from day 10 to day 46 to approximately 85% and increased vertically to 110% after the waterflow change at the 46th test-day. ‘ .muzui Co. Vov hzwmmmn—a >m._.2<0_..=20_m ... F 3:23 mg... om ov ON 0 o . _ . . . o 3:932 l om m v- n n I on T R O M w. .H l 00 A L U M U C co. l on ..meaamw mow com>x0 cop I oo— cozmegmm mow oo>_omo_o .50.. k /# cozoeaaow moo couoezz om— w hzm2hx0 cm I l or I I cozoezmw mow oo>_omm_o .50... ooFI INK cozmcfiow mow cooot.z ; N hzw2k.oom.o .30... / \ cozoeaeow moo concerz n hzm5hx0 coszBmw moo oozomwfi. .80... IIIllll/f cozoegom woo c3952 ..OEPZOO om ov oo ow oow ONF (96) NOllVHanS 8V9 49 Figure 1 demonstrates that the initial onset Of the disease occurred innTreatment 1 after two days of exposure to approximately 117% nitrogen saturation (oxygen 103%) causing a progressive mortality, reaching six percent in the first ten days. Thereafter, the nitrogen levels remained constant, but the oxygen levels diminished gradually to approximately 85%. There was insignificant mortality during a period from day ten tO-day 46. On day 46 the waterflow was changed from 200 to 400 ml/min. The increase in waterflow increased the oxygen saturation level to 110% in Treatment 1. Death began to occur rapidly at that point and continued, reaching 80% at the termination Of the experiment. The oxygen level at the 36th test day was significantly different (P<:0.01) from the level on the 46th test day, (Figure 1). The levels Of nitrogen and total dissolved gas saturation were not significantly different (p).o,01), The injected volume Of air produced by kHP motor at 1.4 atmosphere Of pressure was constant throughout the test, however.1 It is important to note in Figure 1 that the elevated level of nitrogen combined with the increased oxygen levels could have raised supersaturation above the lethal threshold,causing the extensive mortality. Unfortunately, the percent saturations Of nitrogen, oxygen and total diasolved gas were not directly comparable due to variations occurring within groups between determinations. Therefore, the threshold saturation level was not calculated. Figures 2, 3, and 4 show that mortality from GBD did not occur in Treatment 2, 3 and in Controls. Using Chi-square test, there was 6 50 a significant difference between survival rates for the four groups (Treatment 1, 2, 3 and Control) at 1% level of significance (Table 5). Using Bonferroni Chi-square to compare each experimental group with the control, only Treatment 1 was significantly different from control (P<:0.01). The levels Of nitrogen saturation in Treatment 2, 3 and in Controls were approximately at 109, 106, 105%, respectively (Table 6). It is emphasized that these test groups had oxygen levels well below 100%, varying from 85 to 50% saturation. NO 100% nitrogen saturation group was incorporated in the experiment because the normally saturated (100%) well water was not available at the laboratory facili- ties. Observations conducted during the course of the long term experi- ment show that no changes in behavior or survival occurred in the test fish in Treatment 2 and 3 in comparison with the control fish. (Table 5). On the other hand, in Treatment 1, initial mortality occurred within two days. However, the signs associated with the disease were rarely Observed prior to the convulsive stage which occurs a few minutes before death. 2. Signs and Symptoms The GBD syndrome exhibited during the initial mortality during the first ten days of exposure (Treatment 1) and the extensive mortality after the 46th test day (Experiment 3) was similar to that observed in the acute exposure (Experiment 1 and 2). That is, the acute lethality was indistinguishable from the chronic form. Symptoms noted in rainbow trout fry in the Treatment 1 period included loss .of equilibrium, abnormal buoyancy and aimless swimming Table 5. Survival of rainbow trout fry in supersaturated water at 12°C for 60 days. 51 Each test tray contained fifty fish at the beginning of the test. Number of Test Number of surviving Survival trays dead fish fish Total* (%) Treatment 1 Al 45 a 49 8.2 B1 36 45 20.0 C1 34 10 44 22.7 D1 30 15 45 33.3 Treatment 2 A2 0 48 48 100.0 B2 1 46 47 97.9 C2 1 46 47 97.9 D2 0 49 49 100.0 Treatment 3 A3 1 47 48 97.9 B3 0 48 48 100.0 C3 0 50 50 100.0 D3 0 50 50 100.0 Control E1 49 49 100.0 E2 0 48 48 100.0 E3 47 48 97.9 *These fish escaped from the tray during the first day of experiment before the test tray had been covered. Dissolved gas content of the experimental water. Table 6. Oxygen in ' Nitrogen in Totalrdissolved gas in % satur. mean + t.05 SE Determ. % saturation mean-l- t.05 SE % saturation mean+t.05 SE test day NNQOQ’ coupon +I+I+I+L+I coon—am mmcooo cacao—Io —a a-c—o unaware Jdédé HHHHH WNeoe I‘Nh v-Ia-O I-OI-l 115. H F0 1-0 H +I+|+|+l+| I~V\O\F: “1090055 QNONOmQ u-IOQOM O—INOO +I+|+I+|+l EROS? aoooooosox OOOOO H—I—I—o—a HQ’QO‘O HHNo—I HHHHH NTNMv—I—‘t mooaomnn OO\¢OO .—1 I-Iv-S 46 56 Treatment 2 m O‘h- oer—a NIfi-flféV +I+|+|+I+| MONTt—I It" INC—IO I‘lnlnhfi cu~o<3c>oac>c> +l+l+l+l+l "l ". ‘°. ‘1' °2 QVMNOO OOOOO o—I—Io—I—av—I ooxrjmnn u-IOOOO +|+l+l+|+| \fm—o—IN QQQO‘O‘ O‘O‘O‘O‘O‘ 0903000 NMx‘fl-n (“I u :2 8 H 90 O H 5.1 NInIAChN u—INNth HHHHH «carom Gmozrs.‘ T‘Od‘lfiln +|+|+I+L+| axe-rsd'ry NCBION—a eieiaéeiei HHHHH 900‘th INQQ’OO O‘C‘O‘O‘O‘ COntrOl 53 as the convulsion stage commenced. Fish started swimming side and belly-up with violent whirling movements interspersed with periods Of inactivity followed by spasmodic convulsions (the moribund stage), leading to death. Fish were frequently observed to die with the mouth agape and the gills and operculum flared. Gross examination of recently dead and moribund fish showed that in nearly all cases Observed, gas bubbles on the surface Of the fish's body were present. Fish develOped the classic signs Of GBD such as subepithelia emphysema of the head (Plates 1 and 2), external and internal surface Of the Operculum, along the branchiostegal region, inside the mouth, and on the gills and in the fins. The above external signs Observed in this experiment confirm the results of Shirahata (1966), Rucker and Kangas (1974) and Stroud et al. (1975). The gills, in some cases, appeared swollen and were covered with excess mucus. Gas blisters on the margin of one or both Opercula were Observed in most of the affected fish. Gas bubbles were observed in all fins, with the exception of the adipose, and were most commonly observed in the dorsal, caudal anal fins (Plates 3 and 4). Additionally, gas bubbles appeared in the interbranchistegal membrane and the orbits of eyes directly Under the cornea. These signs are similar to those de- scribed by Shirahata (1966) who exposed rainbow trout fry to super- saturated water. It appears that the occurrence Of these signs is associated with the convulsive stage. Evidence of subepithelia gas bubbles on the fish's body prior to that stage was apparent in only a few cases. Macroscopically visible emphysema without rapid Bouin's fixation Plate 1. 54 A photograph Of a 3-month-Old rainbow trout exposed for 48 days in supersaturated water at 115 percent TDGS, showing focal elevation of the epithelium over the caudal margin Of the Operculum and over the skull (arrow). (millimeter scale; 5X) _. magn— Plate 2. 56 A photograph Of a 3-month-Old rainbow trout exposed for 53 days in supersaturated water at 115 percent TDGS, showing the separation of the epithelium.from subcutaneous tissue filled with gas over the base Of the skull (arrow) and at the caudal margin of the Operculum. 5X Plate 2 Plate 3. 58 A photograph of a 3~month—Old rainbow trout exposed for 50 days in supersaturated water at 115 percent TDGS, showing emphysemahmm lesions Of the caudal fin (arrow). 5X n ova—n— 60 Plate 4. A photograph of a 3~month-Old rainbow trout exposed for 50 days in supersaturated water at 115 percent TDGS, showing anphysematous lesions of the anal fin (arrow). 5X .v Ono—n. 62 disappears within a few hours after death, when fish are removed from the supersaturated water. Signs of GBD were reported to have dis- appeared after death (Bouck 1976) and leave the carcass without dia- nostic signs. Approximately ten percent of the fish (Treatment 1) develOped exo- phthalmia which did not necessarily lead to death (Plate 5). Over time, some affected fish that showed popeye suffered loss Of one or both eyes. In a few other fish, the condition of exophthalmia regressed, leaving the eye intact. 'Tests for visual function were not conducted. Gross GBD signs also included periorbital hemorrhage in some affected fish. Commonly in the gills, in the Opercular and branchio- stegal areas, petecheal hemorrhages were observed. Fish appeared to die most frequently during periods of stress such as feeding, gas monitoring, or any manipulation which increased muscular activity. Stroud and Nebeker (1976) also observed that fish more frequently died Of gas embolization shortly after physical stress was induced. In addition, some-fish died without visible external signs. IV. Pathology A. Acute Gas Bubble Disease (Experiment 1 and 2) All fish used in these preliminary experiments were fixed, examined and compared with controls. Histological sections were evaluated and it was concluded that the rapid fixation preserved lesions better than the standard fixation technique, since for laboratory diagnosis Of GBD, macroscopic and microscopic emphysema was well preserved. Acute exposure of experimental fish to supersaturated water (:130%‘ TDGS) caused death within approximately 25 mintues. Therefore, the Plate 5. 63 A photograph of a 3~month-old rainbow trout exposed for 55 days in supersaturated water at 115 percent TDGS. There is a marked unilateral exophthalmia (arrow). 5X m mum—n. 65 lesions were Often less Obvious than those Observed in chronic exposure experiments. The lesions consisted Of external gas blisters in the fins which arose immediately before the onset of convulsions. The most striking pathological changes were observed in the afferent branchial arterioles Of the gill filaments which revealed spaces, suggesting fixed gas embolization. In some specimens, the air space was continuOus with the branching of the ventral aorta. Microscopic examination of the brain of some specimens showed a large space in the skull, usually associated with and surrounding the brain stem. This lesion may account for some mortality in acute GBD through pressurization at the base of the brain;‘ These lesions are considered below, (5- Additional .lesions). *e B. Chronic Gas Bubble Disease (Experiment 3) 1. Introduction Histological sections of the major organs and tissues of rainbow trout fry specimens exposed to long term gas supersaturation were examined in a similar fashion to those described above. Samples were taken of moribund fish throughout the course of the test and at the end of the experiment, to determine the cause Of death and to charac- terize the signs of gas bubble disease that had develOped. Gross, macroscopic and microscopic lesions of the organs were studied to locate involved organs and tissue components. The extent, severity and nature Of the pathologic changes were determined in this manner. In chronic exposure the histopathologic findings in moribund fish were very similar in extent and type to most lesions of the acutely 66 lethal (Experiment 1 and 2) supersaturation trials; however, in those fish surviving chronic exposure and in controls there were additional lesions which were distributed randomly among exposure and control groups. Because the hypothesis predicted a chronic effect, changes were anticipated in various organ systems. Therefore, each treatment group was sampled so that whole fish, viewed with dissecting microscope (macroscopic examination) as well as fixed multiple microscopic sections were surveyed. These examinations revealed lesions which were then selected for additional microscOpic sectioning. It was possible to make gross and microscopic comparisons. The histopathologic results, split between the specimens from 149 moribund fish, and the specimens from 104 fish at termination were all examined grossly. There were 12 moribund fish selected for micro- scopical study along with an equal number of fish from the holding tank. There were 422 termination survivors examined and 104 were sectioned for microscopical study. Lesions were categorized by making a blind comparison among treatment and control fish. Because Of lesions en- 'countered, additional sections Of gills, liver and kidney were made and examined. 2. Subcutaneous Emphysema The most common grossly detectable lesion was subcutaneous tissue emphysema elevating the skin. This lesion took several forms. A ballooned-out epithelium elevating the basal layer of epithelium at the caudal margin of the Operculum and over the skull (Plate 1 and 2) were the most common skin lesions in the chronic study. These lesions 67 occurred in all fish which died. The above confirm the results of Meekin and Turner (1976) who reported that cutaneous gas bubbles were the most common signs in juvenile chinook salmon exposed to gas supersaturated water. Another very common lesion was emphysema Of the caudal fin (Plate 3), dorsal fin, pectoral fin and anal fins (Plate 4). These lesions occurred in all Of the acute supersaturation and the majority Of the moribund, chronically exposed fish. 3. Exopthalmia In 5 of 50 fish in Treatment 1 of the chornic study, there was unilateral exophthalmia (Plates 5 and 6) and one case of bilateral exophthalmia. .Meekin and Turner (1979) reported exOphthalmia in less than 5% Of dead juvenile chinook salmon after exposure to supersaturation. In some affected fish there was darkening of the skin due presumably to the loss of melanophore reactivity to light. On examination of the occular lesion in the severe form, the globe of the eye was prolapsed outward from a point adjacent to the optic disk (Plate 6) resulting in a space forming between the capillary layer Of the choroid and the pigment epithelial layer of the retina. This space occupied upto one half the volume Of the normal globe. No particular inflammatory lesion was associated with the prolapse. The earliest occular lesion detected was that of an emphysematous space within a vascular space of the choroid body Of the eye (Plate 7). Another microscopic lesion, more advanced and presumably the precursor lesion to globe prolapse, was a dissection Of the retina from the choroid with an intact Optic nerve. The dissecting separation resulted in formation of an oval tent of retinal tissue around the Optic nerve, the separation occurred at the 68 Plate 6. A cross section of the head through the eyes of a 3—month- Old rainbow trout exposed for 52 days in supersaturated water .at 115 percent TDGS, showing a macroscopic lesion of severe unilateral exophthalmia (arrow). 18X o mum—n. 70 Plate 7. Detail of the eye of a 3-month-Old rainbow trout exposed for 49 days in supersaturated water at 115 percent TDGS, showing a lesion Of the early fiexophthalmia manifested by the presence of an air space in a vessel Of the choroid gland (arrow). 200x Plate 7 72 disk which served as the only point of remaining attachment (Plate 8) and minor degenerative lesions of the Optic nerve were also observed. In the normal eye (Plate 9) there was an intimate association between . the choroid gland, choroid layer and the pigmented epithelium of the innermost retinal layers. In the affected eye, degenerative vacuoli- zation Of the Optic nerve, musculature, adjacent connective tissue and glandular tissue was evident (Plate 10). 4. Gill Lesions A lesion found in every moribund fish, both after chronic and acute supersaturation treatment, was gas displacement of the blood from the afferent arteriole within the gill filaments. 'The lesion was never recorded in controls. Marsh and Gorham (1905) described gas bubbles in the gill filaments as the most constant and significant macroscOpic lesion. The gas emboli affected arteriole was identified by its anatomical association with the cartilagenous support of the gill arch (Plates 11, 12, 13 and 14) while the efferent arteriole, which was not marked by gas-blood displacement, was identified by anatomical characteris- tics of the fleshy, broad end of the gill filament. In addition, the ventral aorta, which supplies blood in branches into the afferent ar- terioles immediately after leaving the bulbus arteriosus ofthe heart, was occasionally recorded as also having a.lesion of gas-blood dis- placement (Plate 15) while the control sections did not demonstrate such lesions (Plate 16). The findings suggested that the origin of gas was from the heart with build-up in the afferent side of the gill arches. The lesions were histOpathOlogically similar in both acute and chronic treatment fish. 73 Plate 8. Detail of the eye of a 3-month-Old rainbow trout exposed for 52 days in supersaturated water at 115 percent TDGS, showing the precursor lesion to occular prolapse (arrows) in GBD induced “exophthalmia. 200x m cum—n. 75 Plate 9. Detail of the eye of a 3-month-Old rainbow trout from.the control group, showing the normal appearance Of the Optic nerve, choroid and retina. 200x Plate 9 77 Plate 10. Detail of the eye Of a 3—month-old rainbow trout exposed for 52 days in supersaturated water at 115 percent TDGS, showing the degenerative vacuolization of the Optic nerve, adjacent connective and muscle tissue in GBD induced eXOphthalmia (arrow). 200x or man:— Plate 11. 79 A cross section of the gill filaments of a 3-month-Old rain- bow trout exposed for 53 days in supersaturated water at 115 percent TDGS, showing the gas displacement of the blood from the afferent arterioles (straight arrows) and normal blood content of the efferente arterioles (curved arrows). 200x Plate 11 81 Plate 12. A cross section Of the gill filaments of a 3~monthrold rainbow trout from.the control group, showing the appearance of normal afferent (straight arrows) and efferent arterioles (curved arrows). 200x NF wear. 83 Plate 13. Detail of the gill filaments of a 3-month-old rainbow trout exposed for 53 days in supersaturated water at 115 percent TDGS, showing gas displacement of the blood from the afferent arterioles (arrow). 500x Plate 13 85 Plate 14. Detail of the gill filaments of a 3-month-Old rainbow trout from the control group, showing the appearance of normal afferent arterioles (arrow). 500x Plate 14 87 Plate 15. A longitudinal section of the gill arch of a 3—month-Old rainbow trout exposed for 53 days in supersaturated water at 155 percent TDGS, showing the ventral aorta with evidence for gas displacement of blood (arrows) close to the origin of the afferent branchial arteries. 200x Plate 15 89 Plate 16. A longitudinal section of the gill arch of a.3-month-Old rainbow trout from the control group, showing the normal appearance of gill structure and ventral aortic vasculature without gas-blood embolization (arrow). 200x Plate 16 91 5. Additional Lesions In two fish exposed to acute supersaturation conditions, an em- physematous space was apparently fixed within the skull (Plate 17). The spaces surrounded the medulla oblongata and appeared to "compress" the brain. The suspect lesion was visible on sagital and on cross section in one fish. The spaces were apparently continuous with semi- circular canals, and, because it is not possible to prove the presence of gas after fixation, may have been an artifactual because of oblique sectioning of the auditory passages which gave the appearance of emphysematous lesions. Control sections revealed undilated semi-circular canals containingzntamorphous protein fluid (Plate 18). Additional lesions were observed in 70 individual fish from the chronic treatment groups. They consisted of various gill lesions, ex- traordinary liver glycogen accumulation, fatty vacuolization of the liver, possible mineralization of scattered kidney tubules and suspected lymphoid hyperplasia Of the thymus gland associated with gill alterations. All of these lesions were randomly distributed among treatment and control groups with no frequency association with a particular super- saturation exposure. Gill lesions, seen in 21 fish on gross and/or microscopical examination, consisted predominantly of epithelia hyper— plasia. This change was segmental or nodular in two cases, but most Often diffuse and associated with accumulation of mucous. Gill fila- ment blunting and fusion was observed sporadically when sections of whole gill arches were sagitally cut but not frequently when gills were cross sectioned. 92 Plate 17. A sagittal section of the brain Of a 3.5-week-Old rainbow trout exposed to acute GBD, (TDGS _ 130%) showing dilated semi-circular canal surrounding the medulla oblongata and brain stem (arrows). 80X Plate 17 94 Plate 18. A sagittal section of the brain of a 3.5-week-old rainbow trout from the control group, showing the semicircular canal space (arrow), medula oblongata and brain stem. 80X Plate 18 SUMMARY AND CONCLUSION Following the acute case study, the rapid fixation technique for histopathological examination was shown to be useful for laboratory diagnosis of GBD since gas emboli were better preserved than with standard techniques. Therefore, the hypothesis that temperature and time may influence fixatiOn was accepted at the conclusion of Experiments 1 and 2. After evaluating the results of the chronic exposure Of rainbow trout fry to supersaturated water, it was logical to conclude that the ratio of the saturation levels Of oxygen and nitrogen as well as total dissolved gases are critical in inducing GBD. Tables with statistical analyses correlating gas saturation levels with mortality are not presented since the gas levels varied within treatment groups; in.addition, control group nitrogen saturation levels were above.100 percent. Nonetheless, these tests suggest that gas supersaturated water may produce GBD in fish.especially when both nitrogen and oxygen saturation levels are above 100 percent, but it remains unclear exactly what nitro- gen level would have had substantial lethal effect in this experiment assuming oxygen levels were.over 100 percent. Levels of nitrogen satura- tion up to 117% did not produce GBD in rainbow trout fry when oxygen. saturation levels were below 100 percent. During the course Of the test, 96 97 mortality increased drastically when oxygen levels were elevated above saturation. The final percent saturation of oxygen achieved with the change in waterflow'was presumably the result of the difference in partial pressure Of two gases, N2 and 02, and their solubilities in water. It is suggested that the mortality occurred (Figure 1) as an exacerbation of an acute and pathologic gas embolization rather than as a result Of a chronic disease process. Therefore, the hypothesis tested in Experiment 3, which suggested a basic difference in pathogenesis between acutely lethal and chronic GBD, was rejected. The detected lesions in chronic supersaturation exposure, besides the exophthalmia, were similar in acute GBD or were unrelated to treatment, since they were equally frequent in control fish. In summary, the data indicate that an undefined physiological factor, triggered by environmental factors and probably oxygen supersaturation in combination with nitrogen excess in the blood stream, appeared to cause the gas bubble.formation and gas embolization. A gas bubble disease hypothesis which is logical (Bell and Farrel 1972) hypothesizes that violent oxygen unloading, characteristic of some fish.hemoglobin under conditions in which blood is gas saturated, forms small oxygen emboli. These enboli build to form larger enboli under conditions of high.total dissolved gas pressure. Laboratory investigations have shown that fish probably only respond to the level Of oxygen in the.water and blood (Eddy 1971) rather than carbon dioxide levels as do.mammals. Therefore, fish.respond to excess of oxygen levels by slowing the respiratory and circulatory efforts even though.their tissues may be accumulating lactic and carbonic acids. This metabolic precess produces a decrease in the blood pH Bohr effect) which 98 is enhanced by the increase in the partial pressure of carbon dioxide (Root effect). This process could precipitate a violent oxygen unloading from.red blood cells and would form.the oxygen emboli. This process may occur even faster during physical stress, as in feeding or handling, since the increase in muscular activity can raise the partial pressure of carbon dioxide in venous blood from.2.5 to up to 8 mmHg (Holeton 1971a, b; Randall 1975). Histopathologic lesions_were located in the gill arches, choroid gland Of the eye, brain and peripheral tissues. Since each.of theSe areas are associated with acid secreting tissue and may foster accelerated gas exchange from hemoglobin, the lesion distribution suggests that oxygen unloading may play a role. Well water supply is Often supersaturated with.gases. A practice currently used.in.Michigan fish.hatcheries to remedy nitrogen super- saturation is tO increase aeration. However, high oxygen levels in the field have not been assessed with.regard to GBD. This research pre- liminarily reports that water supersaturated with.nitrogen may induce GBD in fishif the levels of oxygen are above 100 percent. It may be recommended that, in situations where maintenance Of oxygen below saturation levels is possible when nitrogen supersaturation is present, reduced oxygen saturation may be a method to reduce mortality. LITERATURE CITED LITERATURE CITED Adams, E. S. and F. G. Towle. 1974. Use of a recompression chamber to alleviate gas bubble disease in coho sac-fry. Prog. Fish Cult. 34: 41. ‘ Anaonymous. 1982. U.S. Department Of the Interior. Fish and Wildlife Service. Quarterly report of progress. April/June, 1982. Bell, T. G. and R. K. Farrel. 1972. Gas bubble disease: Laboratory studies on gas emboli formation and mortality in the steelhead trout. The Hearing on Nitrogen Supersaturation, Subcommittee on Public WOrks, HR, 92nd Congress, May 6, 1972, USGPO 80- 152. Bouck, G. R. 1980. Etiology of gas bubble disease. Trans. Am. Fish. Soc. 109: 703-707. . 1982. Gasometer: An inexpensive device for continuous moni- toring of dissolved gases and supersaturation. Trans. Am. Fish. Soc. 111: 505-516. ' 1976. Supersaturation and fishery observations in selected Alpine Oregon Streams. Pages 37-40 i§_Flickeisen and Schneider (1976). Gas bubble disease. CONF-741033, Technical Information Center, Energy Research and Development Administration, Oak Ridge, Tennessee, USA. Chamberlain, G. W., W. H. Neill, P. A. Romanowsky, and K. Strawn. 1980. Vertical responses of Atlantic Croaker to gas supersaturation and-temperature change. Trans. Am. Fish. Soc. 109: 737-750. Crunkilton, R. L., J. M. Czarnezki, and L. Trial. 1980. Severe gas bubble disease in a warmwater fishery in the.Midwestern United States. Trans. Am. Fish. Soc. 109: 725-733. Dawley, E. M., M. Schiewe, and B. Mbnk. 1976. Effects of long-term.ex- posure to supersaturation Of dissolved atmospheric gases on juvenile chinook salmon and steelhead trout in deep and shallow tank tests. Pages 1-10 in Fickeisen and Schneider. Gas bubble disease. CONF-741033, Technical Information Center, Energy _ Research.and Development Administration, Oak Ridge, Tennessee, USA. '1' /. Dejours, P., J. Armand and G. Verriest. 1968. Carbon dioxide dissociation curves of water and gas exhange of water-breathers. Respiration Physiology. VOl. 5, pp. 23-33. 99 100 Ebel, W. J. 1979. 'Effects of atmospheric gas supersaturation on survival of fish and evaluation of proposed solutions. In United States Army Corps of Engineers. Fifth progress report on fisheries engineering research program 1973-1978. Portland District, Fish and Wildlife Section, Portland, Oregon, USA. Eddy, F. B. 1971. Blood gas relationships in the rainbow trout (Salmo gairdneri). F. Exp. Biol. (1971), 55: 695-711. Egusa, S. 1959. The gas disease of fish due to excess of nitrogen. Journal of the Faculty Of Fisheries and Animal Husbandry, Hiroshima University, 2: 157-182. Embody, G. C. 1934. Relation Of temperature to the incubation period ' of eggs of four species Of trout. Trans. Amer. Fish. Soc. 64: 281-292. . Harvey, H. H. 1974. Gas disease in fishes - a review. Pages 450-485 in W. A. Adams, editor. Chemistry and physics Of aqueus gas solutions. The Electrochemical Society, Princeton, New Jersey, USA Harvey, H. H., and.A. C. Cooper. 1962. Origin and treatment of a supersaturated river water. Int. Pac. Sal. Fish. Comm. Prog. Rep. 9. 19 pp. . Holeton, G. F. 1971a. Oxygen uptake and transport by the rainbow trout during exposure to carbon monoxide. F. Exp. Biol., 54: 239- 254. ' . 1971b. Respiratory and circulatory responses of rainbow trout larvae to carbon monoxide and to hypoxia. F. Exp. Biol., 55: 683-694. Jones, D., and D. H. Lewis. 1976. Gas bubble disease in fry Of channel catfish (Ictalurus punctatus). Prog. Fish. Cult., 38: 41. Marsh, M. C., and F. P. Gorham. 1905. The gas disease in fishes. Report of the United States Bureau of Fisheries (1904): 343-376. Meekin, T. R., and B. R. Turner. 1974. Tolerance of salmonid eggs, , juveniles and squawfish to supersaturated nitrogen. Washington Department of Fisheries Technical Report 12: 78-126. Nebeker, A. V., D. G. Stevens, and J. R. Brett. 1976. Effects of gas supersaturated water on freshwater aquatic invertebrates, Pages 51-65 in Fickeinsen and Schneider. Gas bubble disease. CONF- 741033, Technical Information Center, Energy Research and DevelOpment Administration, Oak Ridge, Tennessee, USA. Nebeker, A. V., D. G. Stevens, and R. K. Stroud. 1976. Effects of air- supersaturated water on adult sockeye salmon (Oncorpynchus nerka). Fish. Res. Board Canada, 33: 2629-2633. 101 Nebeker, A. V., Andros, McCrady, and D. G. Stevens. 1978. Survival Of steelheaditrout (Salmo gairdneri) eggs, embryos, and fry in air-supersaturated water. J. Fish. Res. Board Can., 35:261-264. Newcomb, T. W. 1976. Changes in blood chemistry of juvenile steelhead, Salmo geirdneri, following sublethal exposure to nitrogen supersaturation. Pages 96-100 in Fickeisen and Schneider. Gas bubble diesase. CONF-741033, Technical Information Center, Energy Research and Development Administration, Oak Ridge, Tennessee, USA. Pauley, G. B., and R. E. Nakatani. 1967. Histopathology Of "gas bubble" disease in salmon fingerlings. Fish. Res. Bd. Canada, 25: 867-871. Randall, D. J. 1975. Carbon dioxide excretion and blood pH regulation in fish. Pages 405-418 in.Chemistry and Physics of Aqueous Gas Solutions. The Electrochemical Society, Princeton, N.J. Rucker, R. R. 1972 Gas-bubble disease Of salmonids:’ A critical review. Bur. Sport Fish. Wildl. Tech. Pap. 11 pp. Rucker, R. R., and K. Hodgeboom. 1953. Observations on gas bubble disease of fish. Prog. Fish. Cult. 15: 24-26. Rucker, R. R., and P. H. Kangas. 1974. Effect of nitrogen supersaturated water on coho and chinook salmon. Prog. Fish Cult. 36: 152-156. Shrahata, S. 1966. Experiments on nitrogen gas disease with rainbow trout fry. Transl. by Nat. Mar. Fish. Serv., 1971. Bull. Fresh. Fish. Res. Lab., 15(2): 197-211. Speece, R. E. 1969. U-tube oxygenation for economical saturation of fish hatchery water. Trans. Amer. Fish. Soc., 4: 789-795. Stroud, R. R., G. R. Bouck, and A. V. Nebeker. 1975. Pathology of acute and chronic exposure of salmonid fishes to supersaturated water. Pages 435-449 in W. A. Adams, editor. Chemistry and Physics of Aqueous Gas Solutions. The Electrochemical Society, Princeton, N.J., USA Stroud, R. R., and A. V. Nebeker. 1976. A.study of the pathogenesis of gas bubble disease in steelhead trout (Salmo gairdnepi). Pages 66-71 in Fickeisen and Schneider. Gas bubble disease. CONF-741033 Technical Information Center, Energy Research and Development Administration, Oak Ridge, Tennessee, USA. Weast, R. C. 1980. Handbook of Chemistry and Physics, 6lst edition (1980-1981). CRC Press, Inc. Weitkamp, D. E. 1976. Dissolved gas supersaturation: live cage bio- assays at Rock Island Dam, Washington. Pages 24-36 in Fickeisen and Schneider. Gas bubble disease. CONF-741033. Technical ' Information Center, Energy Research and Development Administration, Oak Ridge, Tennessee, USA. 102 Weitkamp, D. E., and M. Katz. 1980. A review of dissolved gas supersa- turation literature. Trans Am. Fish. Soc. 109: 659-702. Westers, H. 1983. Experience in Michigan with gas supersaturation. Gas Supersaturation Workshop of the Bio-Engineering Section, Amer. Fish. Soc., Milwaukee, WI, July 16, 1983. Westgard, R. L. 1964. .Physical and biological aspects of gas-bubble disease in impounded adult chinook salmon at McNary spawning channel. Trans. of the Amer. Fish. Soc., 93: 306-309. Woodbury, L. A. 1941. A sudden mortality of fishes accompanying a super- saturation of oxygen in Lake Waubesa, Wisconsin. Trans. Am. Fish. VITA Joao Paciano Machado was born in Silvania, Goias, Brasil, on March 9, 1952. He is the son of Joao Vieira and Florinda.Machado. He attended highschool in Goiania, Goias, from 1964 to 1967. He attended Goiania Institute, Goiania, Goias, where he received a Sciences Degree in Biology in 1970. He was acepted by Federal University of Goias at Goiania.where he completed a Bachelor's Degree in Veterinary Medicine in 1976. He worked as Assistant Director of the Rural Extension Service, Department of Agriculture, Itapuranga and Trindade, Goias, Brasil from.1976 to 1979. He was awarded a grant from National Council of.Research.(CNPq), Brasil, to support graduate program at Michigan State University in 1982. ICHIGRN STRTE UNIV. LIBRQRIES ll1111111111NIW11WIllWI1111111111111 31293015913936