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HomeMy WebLinkAboutAPA2131[M]&[R1~£o~~£@@@ Susitna Joint Venture Document Number ~I 3 l Please Return To DOCUMENT CONTROL Temperature Tolerance in Young Pacific Salmon, Genus Oncorhynchusl BY J. R. BRETT • Pacific Biological Station, and Department of Zoology, L""niversity of Toronto (Received for publication Septenzber 19, 1951) Introduction Acknowledgeme.<ts :;\Iaterials :\Iethods Results . Upper limits of temperature tolerance Lower limits oi temperature tolerance Preferrerl temperatures Comparison of temperature resistance Zones of thermal tolerance Discussion and Conclusions Time and temperature CONTENTS Comparison with some other salmonoids Some ecological relations Summary References App~ndix I. Statistical procedure Appendix II. Table:; IV-XIV ABSTRACT , 266 267 267 272 277 277 283 291 293 296 298 298 302 302 305 307 311 315 Lethal limits of high and low temperatures were determined for the young of fiye species of Pacific salmon, the spring (Oncorlr:yncht~s ts!tawytscha), the pink (0. go.~busclta), the sockeye (0. ne•ka), the churn (0. kl'ia) and the coho (0. kisutch). For acclimation temperatures ranging from 5o to 24°C. significan~ rlifferences between sr.:dt:::> in their resistance to high temperatures was obtained. The spring and cono were most resistant. The pink and churn salmon were least resistant, and the sockeye was distinguishable from the latter two by greater resistance for pt'Olonged exposure to high temperatures. ;{o species could tolerate temperatures exceeding 25.1 °C. when exposed for one week. A fanning~out of the opercula was shown to be directly correlated with the onset of death from a low temperature. By use of this criterion mixed le~hal effects at low itm~peraturcs were demonstra- ted and found to be influenced by the size of the fish and by the salinity of the watet'. None of the species could with:;tand temperatures lower than 4°C. when acclimated to 20°C. and above. When taken from holding troughs as low as 5°C., coho and sockeye could not tolerate long exposure (four daysJ to 0°C. -·--- tBased upon portions of a thesis accepted by the Faculty of Graduate Studies, t"niversity of To1·onto, in partial fulfilment of the requirements for the degree of Doetor of Philosophy. fi. F.tSH. RL~S. ao··.· C.-\~ .• 9 tU), Hl5;} [}rinted in Canada. :J 265 - : 1' ·~ J .. :.:.··,. t~ . . . f ~ ' I l I I \ I I l: I .:_·, 266 · In a vertical gradient little difference in preferred tE'mperature was observed, either between spectes or for different acclimation temperatures. The 12° to l4°C. stratum was the region of great~st concentration. Specific <!iifferences in temperature responses are in keeping with taxonomic and ecological diEtinctions. INTRODUCTION No ACTIVITY of an animal escapes the effect of temperature. Distribution, develop- ment, propagation and mere existence, each is influenced strongly in some manner by temperature. This influence must · 'e met and surmounted eitht•r through resistance or adaptation, external avoidance or. internal control. It is known that the upper and lower limits of temperature-tolerance in fish arc extended through both adaptation and resistanc~ ~Fry, 1947a), and the varying degrees of these t\\'O attributes sepa~ately and collectively distinguish :he species in this respect. By conducting experiments on tolerance to high and to low temperatures among the young of the five North American species of Pacific salmon, the relative abilities of these species to cope w::h extremes of temperature have been des(.ribed in the following analysis. Among earlier experiments, interest in the ability of fish to survive tempera- tures in the region of the freezing point of water and slightly below was expres:ed by Regnard (1895). In 1899, Maurel and Lagriffe while invetJcigating both upper and lower levels of temperature-tolerance, chiefly in fresh-water fish, concluded that these species were better adapted for resisting low than high temperatures. Later investigators, dealing mainly with mort;:~lity froo. high temperatures, ex- pressed the resistance in t:erms of the tem;_:.,erature reat:l.::d before death when heated at a constant rate (Huntsman and Sparks, 1924)r or averaged either the times to death (Loeb and \Vasteneys, 1912) or the number of fish dead following a given exposure (Hathaway, 1927) at various constant temperatures. This quantitative expression of temperature-tolerance has been developed to provide a more inclusive treatment, borrowing from the methods of pharmacological ·procedure ~oncerning dosage-mortality (Fry er; al., 1946). The phe~1omenon of thermal adaptation in relation to previous temperature history provides the organism with greater scope for environmentdl experience. The term "acclimation" has been used. to describe this effec~, although "acclima· tization" is apparently synonomous (Doudoroff, 1942; Heilbrunn, 1943; Brett. :!~44). The importance of temperature-acclimation in nature and in experimental work has been stressed with significant emphasis (Doudoroff, 1945; Fry, 1947a), By working systematically with ,different temperature-acclimations the variou.s levels of both upper and lower thermal tolerance can be determined within sufft- cient statistical limits to permit accurate prediction. The development of precise methods of physiologtcal me:asurement has set the stage for physiological description. The close taxonomic relation of the Pacific salmons, genus Ortcorhynclw.s, (l'viilne, 1948) coupled with faidy distinct ecofogical habits, provides interest Hl the affinities whkh might be revealed by a rigorous analysis of their temperature· tolerances. Similar work on other ::salmonoids is mounting (Fry et al,. 19·10; - ,. ~--.. 4 ! Fry, 1947b~ attributes on the role of t animals \Vill l I am gr~e both directe · · wide variety · • ~l has been the ,. The matli of Dr. D. B. ~· The expel· logy whi. ch i.s .. ·. of Lands and ! ment of Lant labor.a tory a1.1" fish by 1\ilessti measnre to th~ fhe salmt Pacific Biologq of Fisheries, \\ tional shipmet3 The Fish' extended leav . tance. It is a SouRcE The five America were Each lot , insulated box,·· Successful shi TAULE I. The Species l. Spring 2. Pink 3. Sockeye 4. Chum 5. Coho I [ I t I I l \ I t l I , I l (lrf,! :5-ri~ r fl I !'Cfla ~ low 267 Fry, 1947b; Graham, 1949). vVith the gradual recording of such physiological attributes on a comparable basis within and between taxonomic groups of fishes, the role of temperature in the ecology, and possibly in the evolution, of these animals will become increasingly apparent. ACKNOWLEDGMENTS I am greatly indebted to Dr. F. E. J. Fry who has for a number of years both directed and stimulated research on temperature relations in fish. His wide variety of interests and knowledge in the problems of experimental biology has been the source <:t <t wealth of suggestions and .?.. constant inspiration. The rnathematica1 analysis of the data has bee.~: •;.m.der the helpful guidance of Dr. D. B. DeLury of the Ontario Research Foundation. The experiments were conducted in the Laboratory for Experimental Limno- logy which is operated jointly by the University of Toronto and the Department of Lands -nd Forests, located at the Southern Research Station of the Depart- ment of Lar'ds and Forests, 1\llaple, Ontario. The excellent facilities of the laboratory and the attention devoted to feeding and maintaining the stocks of fish by 1t1essrs. D. Cucin, G. 'Stolfa and \V. Sanderson contributed in a large measure to the successful execution of the research. The salmon eggs were obtained through the courtesy of i\1r. F. );eave, Pacific Biological Station, British Columbia, and of Mr. C. H. Ellis, Department of Fisheries, \Vashington. They have each responded to urgent pleas for addi- tional shipments when transp~rtation problems resulted in minor catastrophies. The Fisheries Research Board of Canada has not only seen fit to grant extended leave of absence to the author, but has al!1o given him financial assis- tance. It is a pleasure to acknowledge this support. l\IATERTALS SOURCE The five species of Pacific salmon common to the west coast of Xorth America wete obtained as eyed eggs from three hatcheries (Table I). Each lot of approximately two thousand eggs was shipped in a fiberglass- insulated box, pa~ked with ice and perforated to permit air exchange for the eggs. Successful shipment by air was possible if not more than thirty-six to forty-eight TABLE I. The hatchery locations and-dates of fertiliz.ng, shipping~. J 50 per cent hatch for the five species o(Pacific salmon. Species Hatchery Fertilized Shipped 50% Hatch - 1. Spring Dungeness, \Vash. 30/8/49 20/10/4H . 9/11/40 2. Pink " .. 28/9/49 7/12/49 12/12/49 a. Sockeye Issaquah, " l0/10/·19 25/11/411 6/1/50 4. Chum ~ile Creek, B.C. 28/10/-19 7/1/50 4/2i50 5. Coho " " " 14/11/49 7/1/5C 27jl/50 ----, -·- - 0 'J '· e~_ '"' '~' l " .. • l ' I ' -~ I . 1 ... ·. ; :: ''l II :; 11' i i t t !' l f l l l I ,), I , I I l \ I \ \ '' L f I () 2{)7 Fry, 1947b; Graham; 1949). \Vith the gradual recording of such physiological attributes on a comparable basis within and between taxonomic groups of fishes, the role of temperature in the ecology, and possibly in the evolution, of these animals will become increasingly apparent. ACKNOWLEDGMENTS I am greatly indebted to Dr. F. E. J. Fry who has for a number of years both directed and stimulated research on temperature relations in fish. His wide variety of interests and knowledge in the problems of expel·imental biology has been the source of a wealth of suggestions and a constant inspiration. · The mathematical analysis of the data has been under the helpful guidance of Dr. D. B. DeLury of the Ontario Research Foundation. The experiments \Vere conducted in the Laboratory for Experimental Limno- log-; which is operated jointly by the University of Toronto and the Department of Lands and Forests, located at the Southern Research Station of the Depart- ment of lands and Forests, lVIaple, Ontario. The excellent facilities of the laboratory and the attention devoted to feeding and maintaining the stocks of fish by lVIessrs. D. Cucin, G. 'Stolfa and \V. Sanderson contributed in a large measure to the succPssful execution of the research. The salmon eggs \vere obtained through the courtesy of :VIr. F. ~eave, Pacific Biological Station, British Columbia, and of Mr. C. H. Ellis, Department of Fisheries, \Vashington. They hc:ve each responded to urgent pleas for addi- tional shipments when transp~rtation problem.., resulted in minor catastrophies. The Fisheries Research Board of Canada has not only seen fit to grant extended leave of ahsence to the author, but has also given him financial assis- tance. It is a pleasure to acknowledge this support. MATERIALS SOURCE The five speCies of Pacific salmon common to the west coast of X orth America were obtained as eyed eggs fro-n three hatcheries \Table I). Each lot of approximately two thousand eggs was shipped in a fiberglass- insulated box, packed with ice and perforated to permit air exchange for the eggs. Successful shipment by air was possible if not more than thirty-six to forty-eight TABLE I. The hatchery locations and-dates of fertilizing, shipping and 50 per cent hatch for the five species o(Pacific salmon. Species Hatchery Fertilized Shipped 50~:·(; Hatch - 1. Spring Dungeness, \\'ash. 30/8/49 20/10/-!!> 9/11/49 . 2. Pink ,, II 28/9/49 7/12/49 12/12/49 3. Sockeye Issaquah, lt 10/10/-19 25/11/49 6/1/50 4. Chum ~ile Creek, B.C. 28/10/49 7/1/50 4/2/50 5. Coho " II " 14/11/49 7/1/50 27/1/50 . J-' ~-·~-·----,---_,.-~--- J _ _..r.~lllll-tibUIJiidiiBillllM4..._,;,,.,. . ........ . i I I. I ~ L ' -~. 268 hours elapsed between packing and receiving . .An initial mortality not exceerling- 2 per cent inevitably followed the handling necest:itated by shipment. \Yith thi~ excep..:ion, egg losses from other causes were exceptio!lally 1ow in all cases where subsequent experiments were performed. FEEDING AND CARE OF YOUNG The transition stage from alevin to free-swimming, feeding fry is a precarious one for young salmonids. The habit of feeding must be d·~veloped and encouraged, usually by frequent presentation of small particles of food. By directing a jet of water into a small aluminum screened basket containing finely groum' heef or hog livet=, adequate dispersal of thP food over periods of 15 to 20 minut~3, four times daily, was achieved. This routine was maintained for the first month of feeding, followed by reduction in feeding frequency and n. change of diet, mainly in accordance with fish-cultural procedures for salmon currently practised in \Vashington State (Burrows, no date). The diet selected was a slight modification of one reported by h~l.is (1948) which had been found to give best growth and least mortality for young spring salmon when tested on a variety of diets. .\ mixture of 50 per cent beef or hog liver together with -!:8 rer cent grourtd '1fish- pack" (haddock and cod fillet waste) and ) per cent yeast was provided, up to the second month, followed by a reduction in liver w 30 per cent for the balance of the experimental period. :Mortality in the stock tanks with a constaq_t temperature of 8.8° ± 0. r;o was virtually negligible. No prophylactic treatments were introduced. At higher temperatures, 20°C. and above, up to 5 per cent mortality \vas observed in all species, and irrfrequent treatments (t\vo to tlu·ee times per month) with a 1 :4,000 solution of Roccal were applied (Burrows). Two cases where disease became significant '"ere encountered; one with five-month-old chum salmon, from a single tank, which necessitated discarding the remaining fish as well as one series of obviously discordant data; the other, with three-month-old sockeye, raised by stages to 24°C. and apparently incapable of deriving adequate nutrition from their diet at such an elevated temperature. The pH of the well water supply~ng the laboratory was 7.3 with total solids amounting to 254.3 parts per million (Table II). RETAINING TROUGHS The retaining troughs were each supplied with running water tapped from bot-and cold-water sources of relatively constant temperature and pressure. Adjustment of these with regular inspection permitted setting the temperature of a trough (above 9°C.) to within ± 0.1 °C. of any desired temperature. esunllr two, sometimes three, sp~cies were cultured in a divided trough. P.elmv 9.°C. a refrigeration unit reduced the temperature in a single holding tank in which the five species were retained separately in cages of fine aluminum w.ire screening. Thus, different levels of temperature-acclimation were ·readily obtained with a high degree of accuracy and constn,ney. -· I t l t t l t . II SIZE A~D A~\ l.rniforn~~· was maintain. .tt th<',' same t ensure abu nd subsequent 1~ to specific di~ The tim~ :f ~.about two 11. tional two t<] The knowledl in their firc;;t i •r"ges. j _J ~ i " I 5 I l l ~ L I l 1 ;~~ . · ·~-j J .. . . \ . _ .. ~-t'' : .. ~ ... TAEi:..E ll. ::\1\neral constituent-: of the water supply used in these experiments, as reported by the Chief Provincial Analyst, Sept. 24:, 1947. Total solids _-\lkalinity Total hardness Iron and Aluminum (o:\ides) Iron Calcium :\Iagnesium Potassium and Sodium Sulphates Chlorides *As carbonate of lime. parts per million 25±.3 202.0* 1!;';1. 0* 4. 7 0.2 89.5 14.1 36.8 45.1 13.0 269 SIZE AND AGE OF EXPER~:-tENTAL FISH Uniformity of conditions in every feature of tne history of the young fish was maintained as far as possible. Keerir1!!, them in the same or similar troughs, at the same temperature, and presentinf ·~ue same diet in sufficient quantity to ensure abundance, were the first precautions. The variations in response to subsequent high or low temperatures might then be considered as attributable to specific differences only. The time for commencing experiments was set at thr. -e months after hatching (about two months after f~eding commenced), and the"1 continued for an addi- tional two to three months 1 The fish from highGr temperatures were used first. The knO\vledge that the young chum and pink salmen move to salt water early in their first year motivated making comparison of the species i~., the very you·Jg stages. TABLE III. ~lean fork-lengths, weight" and agee of the salraon fry used in temperature-tolerr=~.::e tests. Species Spring Pink Sockeye Chum Coho Spring Sockeye Ch\tm Coho Lenglh (em.) W~ight (g.) Cpper temperature tolerance 4.44 ±0.40 3.81 :±0.29 4.49 ± 0.84: 5.44 ± 0.89 4. 78 ± 0.60 1.03 ± 0.27 o.zo ± 0.15 0.87 ± 0.45 1..62 ± 1.03 1.37 ± 0.62 Lower temperature :tolerance 4. 72 ± 0.4~ 4.50 ± 0.53 5.09 ± 0.51 4.83 ± 0.45 Age (months) 3.6 3.~ 4.7 4.0 5.2 7.7 5.8 5.2 5.! .. ~ ,. I h l j t ! ! l l ~ r l I L .~ ~~ \ ; \ ' I l ! ! I I f r. ·'• I· tU ') , I··. '··. " ;. ' . \ -= 270 In Table III mean fork-lengths and "-eights (vrith standard deviations) and the average age from hatching of all samples used in the temperature-toieranre tests are compiled. No weights are included tor the fish used in low-temperature tests. These samples \Yere not removed from the lethal tanks until some tilile after death. and water absorption had affecten thtir weight. LETHAL BATHS The lethal baths, six,in number and measuring 22 inches square by 11 inches deep (Figure 1) were each constructed of galvanized iron coated inside with aluminum paint and adapted for use in either upper or lower thermal-tolerance tests. The addition of complete insulation with fiberglass of one-inch thickness was of value in reducing temperature variation to a minimrm. Thermostatically controlled, 120-watt heater-coils in pyrex tubing count~rbalanced a steady loss of heat, mainly from aerat;0n and from aver} slmv exchange of wat;.:;rf equal to the volume of the tank every twenty-four hours. In the low-temperature Iethals the heat loss was augmented by the additibn of a layer of crushed ice, partitioned off on three sidt.' of the tank by a removable galvanized iron sheet. A standardized r .Gl"RE 1. Apparatus for determining upper and lower le..:hal tempemtures being assembled. Two units, one with four, th"! other with two lethal baths are shown ·vith connections for thermal control, aerattion and water exchange. (Photograph by Mr. W. P. I<ice.J never high 1 <:oncernmg- variously • :~n inches h t ivcly. Pla romplete t the bottom hol~, the F~GURE 2, lir'qt:!r Jgw il I , I ! ~ r \" \, I. l \' I l I I l I I \ 15 r l \ f~L, \ I \ ! I ~~ "\' ·:ions) and ~tolnrance <npf ature · nine time 271 thermome,er calibrated in intervals of 0.1°C. was used throughout all experi- ments. Ofttn no ~etectable change in the tank temperatures was apparent and, in general, variation did not exceed a range of more than 0.1 °C. from any set level. Under maximum loads of fish per tank (40) oxygen concentrations ,.,ere nevt~" reduced below 93 per cent saturation (5.24 cc. 0:/L, 26.5°C.) in tests on high lethals, and not below 81 per cent (6.91 cc. 02/1., G.0°C.) in low-lethal experiments. PREFERRED-TEMPER.:\..TURE TA~KS Two preferred-temperature tanks were used in a limited series of experiments concerning the .region most frequented in a vertical temperature gradient by variously acclimated salmon fry. These tanks, illustrated in Figure 2, stand 36 inches high, with length and width measurements of 36 and 20 inches respec- tively. Plate glass facings held in angle iron edging and bolted to the main frame complete the outer structure. \Vater, usually of low temperature, is introduced at the bottom through a metal tube perforated uniformly over its length with small holes, the displaced \Vater being drained off at the top. A coiled copper tube FrGURE 2. A preferendum tank davided into ten tells by white cord for recording of fish positions Under low illumination. Thermometers are situated in the front nght of f'ach cell. A faint outline -of the coiled copper tube for carrying hct water is visible within the tank. A~~~Tf'lllllt.Mif ··••• t 4( • ifr.lt JII ~ 1 r., I ' . ' ' 272 forming-a closed system conducts hot water in a downward spiral around the inside p,~riphery of the tank in contradirection t\) the rising "cold fro!lt". By adjusting the temperature and rate of flow of the introduced cold water as well as that of the hot water ·within the copper coil, any desired tempera cure gradient can be obtained. l\IETHODS The approach to the problem of describing the temperature-tolerance of young Pacific salmon has been to hold samples of each species at different non- lethal temperatures but otherwise similar conditions. These variously accl~mated samples were later tested for their tolerance to high and to low temperatures, ranging from rapidly lethal to sublethal levels. The data were treated graphically or mathematically to distinguish such differences as might occur. The application of these methods are considered below. ..:\CCLUlATION As early as 1895 Davenport and Castle reported on the "acclimatization oi organisms to high temperature", and threads of this principle have been variously w·oven around the theme of temperature relations in fish by Loeb and \.Vasteneys (1912), Hathaway (1927), Binet and l\Iorin(l934), Sumner and Doudoroff (1938), and more generally by \Veigmann (1929, 1930, 1936), Ogle and ~Iills (1933), Heilbrunn (1943), and others. Yet a great deal of experimental \Vork has been done \vithout adequate regard for the conditioning effects of temperature in the past-history of animals. A study of changes in heat-tolerance for the goldfish, Carassius auratus, from both low (4°C.) and moderately high (20°C.) temperature- acclimations (Brett, 1946) led to the general conclusion that rate of acclimation was related to metabolic rate. Thus, at low temperatures, acclimation proceeded at a slow rate but incre:tsed to a very rapid rate at high temperatures, probably in geometric progression. Conversely the loss of acclimation to any level of tem- perature was a comparatively slow process under most conditions. The thermal history for the egg, alevin and early fry stage for each of the salmon species was 8.8°C. with very little variation. \Vhen the fry were about two months old the process of moving ther::-t through a series of temperature- acclimations was initiated. The ·minimttm standards set for acclimation from the holding-trough temperature of 8.8°C. to any one of the following temperatures were: 'I' -oc"" 0 i) • To l0°C. To 20°C. To 23°C.(or 2-!°C.) 4 weeks at 5°C. 3 weeks at l0°C'. 3 weeks at l;)°C. 1 week at 15°C.t 2 weeks at 20°C. 1 week at l5°C., 1 week at 20°C., 1 week at 23"~"' Repetition of sQme of the upper Iethal~t:emperature experiments after one and even five months' lapse of time without further change in acclimation gave no significant change in heat-tolerance. l I,'' \ . The fi inclicatcd the hold in fullv cultu , ~ i\ they prov" .tcdimatio live specie .. next ;n linl lethal-tern 2~°C. shO\\. after prolo, divergent ! longed exo · tcmperat~q. more resis~f ..• about 40 1\\ obtained ii\: ,. though at ;t1 <lhnost coni quentlY, thl dnun ;nd 4 11rovidc co {'PPER LE ,u;sociates with the m which prod o( the data HH4; Oou tracing the temperatur · tS necessary ' Each l o.,)°C. diffe chosen by p~ non-lethal t, by dosP. ins plotted gra of an asym various tem fry from 10 1937) that ' .. I l \- l \ I L ., J k r l l -~-1- fi . ' .. : .nij: f he_· ~ · t" · ·B,· ' . ;_,, ... ;IS well ~ . 'ad;<>nt ' f . ' ~ ·; ' .: . . ·, . u .. .. I t \ 273 The first species to be investigated was the spring salmon. Preliminary tests indicated that a maximum acclimation temperature without significant loss in the holding troughs was close to 24°C. This was later substantiated by success- fully culturing over 200 young spring salmon at 2·eC., at which temperature they proved to be very active and to be good feeders, but had reduced growth rate when compared with groups from lower temperatures, particularly those from 15°C. A level of 2-±°C. was introduced, therefore, as the highest standard acclimation temperature for all species. By chance one of the most hardy of the five species had been used to set the standard for the others. The pink salmon, next in line by age for progressive acclimation to 24°C., fed poorly. Immediate lethal-temperature experiments following minimum acclimation standards for 24°C. showed a breaking away fro~ the usuaf temperature-time mortality curve after prolonged exposure to a temperature of 24.5°C. Extrapolation of this divergent trend indicated that 2-!°C. bordered on 50 per cent lethality for pro- longed exposure. Evidence of the unsatisfactory nature of such a high acclimation temperature was convincing in the sockeye fry. The latter species, while apparently lTl.Ore resistant than the pink salmon, showed a complete aversion to feeding in about 40 per cent of cases which later appe.:'lred as typical "pin heads", often obtained in hatcheries when young fish do not develop the feeding habit (al- though at an equable temperature). The growth of the remainder was curtailed ~lmost completely and their activity was quite apparently reduced. Conse- quently, the acclimation temperature was lowered to 23°C. for the sockeye, the chum and the coho. Insufficient numbers of spring and pink salmon remained to provide comparison at the ne\v acclimation level. r·PPER LETHAL TE11:PERATURE The method of lethal-temperature determination as conducted by· Fry and associates has remained basically consistent from its inception (Brett, 1941), with the marked exception of the duration of exposure to given temperatures which produce some but not complete mor.tality. The analysis and interpretation of the data have changed and expanded considerably (see Fry et al, 1942; Brett, 1944; Doudoroff, 19-!5; Fry et al, 19-±G; Fry, 1947a; Hart, 1947, HH9). \Vithout tracing the history of these changes, a discussion of the present treatment of temperature data and the current terminology used to describe the-observations IS necessary .. Each lethal bath is regulated to a constant t~r.tperatur~:; aimost exactly 0.5°C. different from the temperature of an adjacent bath and appropriately chosen by preliminary tests to span the conditions from rapid to slO\v, partial, or non-lethal temperature effects. Records of the times to death for all fish are kept by close inspection. These latter have been called the resistance tint.es which, if plotted graphically on normaL axes in order of occurrence, take on the appearance of an asymmetrical S-curve. A series of such curves can be plotted, inc:luding the various temperatures investigated, as has been done for a sample of spring salmon fry from l0°C. acclimation in Figure 3a. It has been demonstrated (Bliss, 1935a, 1937) that many dosage-mortality curves can be resolved into straight-tine ;{ l . ~ .! '.· ~ ,. ~ 'l ~·. ,, ' ~ ( i ~ "\. !' _ ... ., :; ~ :1 •.. 1 - \f .l D I •' !i l l ! r .ll. ·u '' ll • j I .. 1 1 .• .! ll 'J .. 274 relations if the proper derivative of time is applied and the variation of · response is normally distributed within the sample. By converting the axes into probability units as one variable (order of death) and logarithm of time as the other variable (time to death) a linear progression of points is frequently obtained (Figure 3b). The application of this principle to lethal··temperature experiments with fish was shown to be quite appropriate by Fry et al. (1S46). The hundreds of observations which have since been made on many species of fish (Hart, 19-1-7. 1949; present paper) and found to adhere closely to the above interpretation have added convincingly to the validity of the relation. The normalitv of the distributbn permits application of standarrl. statistical treatments. In ~ddition the mean, median and mode, all coincide in a normal distribution, so the descrip- tive value of the single figure (50 per cent puint) is evident. From each lethal-temperature experiment a series of median resistance times may be plotted for the corresponding levels of temperature, in the manner of Figure 4a. It is apparent that for every state of acclimation the possibility of a series of such points exists. Thus, an overall picture of the effect of temperature can be constructed. Conveniently enough these curves, in the case of high- temperature tolerance, can be resoln~d into straight liaes by using the logarithm of time against temperature (Figure 4/;1). A distinct break in the semilogarithmic ·plot, not otherwise evident, occurs at a progressively earlier point of time in the lines for lower levels of acclimatitJn (usually below 20° to 15°C. for Pacific salmon). The discovery of thi8 break (Fry et al., 1946) and its variable occurrence with acclimation was most significant and has constituted the main difference in experimental procedure from that of earlier investigations. The definition of lethal temperature h3.s been that temperature at w·hich 50 per cent of the population is dead after inde.finite exposure. The stumbling block in the past has been the duration of the experimental test. Doudoroff (1945) questioned the 14-hour p~riod used by Fry et al. (1942) and shortened to 12 hours by Brett (1944). The answer, as indicated above, was provided when breaks in the resistance time-temperature relations showed that mortality from temperature as a primary cause had ceased. The duration, even as long as the seven-day period used for Pacific salmC':l should be govern~d by this factor since it varies for different species and different acclimations, As long as the resistance times continue to be finite the fish arc considered to b~ in a zvne of resisfa1tce. Beyond this lies the zone of tolera11ce (Fry, 1947a). At one acclimation there are any number of resistance times but only one lethal temperature. To distinguish indices derived from high-and low-temper;l- ture experiments the terms ·upper and lower lethal tem~eratures are applit..J respectively. LOWER LETHAL TEMPERATURE Temperatures distributed from 0°C. (0.1 o ± 0.1 °C.) to 7°C. at one degree intervals were used in lower lethal-temperature determinations. Olle or tv.to in· stances of experiments at fractional degrees are ~eportedf but the use of 20 fish per tank fmm a limited total sample preclwded crrrying the investigation to a finer point. - ~ PM .p from high cessation of romplete loss Often no lethal-tern from a large t.•quabh~ h:u been ~ample of I 042; Brett, operations in Close in death the rhilled fish reveries, a co highly signi correlation r almost enti long, 3 inches sa.nples for test tempera rt>gardless of taitwr was container mort ali tics, (similar to u pattern wh •1ny further experiments line of I l I !' l 1' l I l I L r- 1 l ( I .. _,......_ _ _.....-..n ....... ~-----------·--. ~c 275 There seems to be relatively little trouble in deciding when a fish has died from high temperature, except in a few instances (Brett, 1944; Hart, 1949). A cessation of rec;:piratory movements and muscular contraction accompanied by romplete loss of response to stimuli have been regarded as quite decisive criteria. Often no reference concerning such end points is included in reports on upper lethal-temperature experiments. A check on 180 Pacific salmor., including all species, by removing the fish to a lower temperature immediately after "death'' was recorded, resulted in no recoveries. On the other hand the depressing effects of low temperatures, producing a type of "suspended animation", have been the source of considerable trouble in establishing satisfactory criteria of death from this cause. Usually groups of fish at a single low temperature have been removed from a large sample and tested for mortality by immersion in water of a more equable temperature, the recoveries being noted over the first 24 hours. This has been performed at intervals throughout the ~xperiment, or, when only a small sample of fish was available, at the end of a given exposure time (Fry et al., 1942; Brett, 1944). The lack of a more direct criterion of death has restricted operations in this field. Close inspection of Pacific salmon revealed that with the approach of cold- death the characteristically immobile and closely compressed opercula of the chilled fish commence to fan out perceptibly. By systematically recording this symptom before removal of each sample to a testing tank (at l2°C.) for re- coveries, a comparison of the ''predicted" and "actual" mortalities was made. A highly significant correlation between the two was obtained (coefficient of correlation r = + .90, P.o1 = .37), the cases o non-agreement being scattered almost entirely on the side of greater "actual'' mortality. Unpredicted recovery was virtually non-existent. Consequently, the resistance times could be tabluated from direct observation of the fish in the lethal baths as in the upper lethal experiments, and the median resistance times plotted for different degrees of low temperature, a system hitherto not em?loyed in low temperature work. In practice, lots of ten fish 'vere placed in small plastic cylinders (6 inches long. 3 inches in diameter) capped at either end with plastic screening. Two such samples for each species from a given acclimation were inspected at each of the test temperatures. vVhen the number of predicted dead in the first cylinder- regardless of the number dead in the second, had reached 50 per cent, the con, tainer was removed to running water at l2°C. The treatment of the second container varied in order to test the prediction value over a gre:ater range of mortalities, but usually contained estimated deaths of between 50 and 100 per cent. The number of actual dead was recorded 24 hours later and excluded all fish shmving perceptible activity--rarely a questionable category. The median resistance times, determined by plotting the order of death (similar to upper temperature· resistance), when transposed to a graph follow a pattern which has not been possible to convert into a more convenient form by any further resolution of the data. A solution [or the problem of duration in the experiments on effects of high mperature was presented. Following the same ~ line of reasoning, though not <-apparent in this case, a flattening-out of the curves to become asymptopic with the time axis. is indicative of continued, pro- - \ l I l l r t r I l I r r . . longed survival of the sample under the corresponding temperature conditions. This latter inflection occurs by at least 5,000 minutes (three and a half days) in practic::tlly all cases, and considerably earlier for the higher acclimations. Conse- quently, a limit of 5,500 minutes was set for the duration of alllow-temnerature .. work. A further use of the lethal temperature to delineate the biokinetic range of fishes has been illustrated by the construction of a trapezium relating upper and lower lethal temperatures to acclimation tempet·ature (Fry et al., 19-i2, and later references). For every stage of acclimation there is a corresponding lethal tem- perature. In the upper temperature region the lethal and acclimation tempera- . tures approach each other, finally providing an ultimate upper lethal temperature (Fry et al., 1946) beyond 'vhich no extension of temperature-tolerance is possible for the species as we kno·w it. Such relations for the Pacific salmon have be• n illustrated in Figures 20 to 2-1. PREFERRED TEMPERATURE The specific aim of the investigation was to work out in some detail the limits of tolerance; it was also possible to carry out research on temperature selection but of a preliminary nature and consequently presented as such. l\rieasurements of the aggregatio~. of fish in horizontal temperature gradients have been conducted effectively by Doudoroff (1938) and Sullivan (19-HJ). Empfiasis must be placed on the horizontal nature of these gradients since vertical gradients were employed for the Pacific salmon. One highly significant difference is apparent, namely, that a gravity gradient is inextricably involved in a vertical tank. It has been customary to reduce as far as possible all interfering factors when recording the responses of an organism to a gradient of a given identity. The methods employed in the present instance were to habituate ten previously acclimated fry to feeding freely in the preferendum tank for one week at the same acclimation temperature. No other control was instituted. Lh·c Daphnia regularly introduced with finely ground food, and slowly swept around the tank by ~urrents produced through .'.eration, served to scatter the ftsh in an irregular manner throughout the tank, Lighting during the haLituation period was from overhead 1.10-watt bt.tlbs and from sunlight through side windows (Figure 2). . On the day of an experiment, feeding was reduced (excessively fed fish tencl to sink to the bottom when inactive) and only between 10 p.m. and midni~~t were observation'; on distribution in a temperature gradient recorded. At thts time lighting, sufficient only to re;,:-ord positi\ms accurately, was produced by two 3-candle-power sources, placed 35 to 40 inches on either side of a middle point of the tank Any defi!nce of territory which had been exhibited under fu!l illumination was never displayed in the gr£atly reduced lighting of an exp:n· · ment. The distribution in various thermal gradients was then noted by counung the number in each cell of known temperature. .. in rcspon These the five .txes, a acdima during The 1 tTable- \':1:.; been mortalit) The the lcvell par~\llel ~\·rk·s of that for <ttT\ima times for n-sultcd (•quailed st•rvc><l r by Fry or lesser !',\)C<'lCS ll·ngth ~ .wt·limat 1Co mcnt of 1 L I I I l l i r L r . ! I r I ' r r ' : A •• ':' u n I) 1: i I r ..... , 277 STATISTICAL TREAT'.IENT The various sources of variability in response to extremes of temperature and the statistical treatment of the data are presented in Appendix I. ::\'Iost of the calculations were made by the method of analysis of variance.1 RESULTS The actual levels of tolerance to extremes of temperature and the differences in response among the five species have constituted the main theme of study. These are presented by dealing with one aspect of temperature and considering the five species collectively under each heading. "CPPER LUHTS {,tF TEMPERA.TURE TOLERANCE SPRING SAJ ... ~ION. Upper limits of temperature-tolerance include the resistance times and lethal temperatures for each acclimation. A typical series of mortality times at different test temperatures has been depicted in Figure 3a on normal axes, and on probit and logarithmic axes in Figure 39 for young spring salmon acclimated to l0°C. At 24.0°C. and below no deaths were recorded for that sample during the 10,000-minute (one week) duration of the experiment (Table IV). The lethal temperature therefore lies somewhere between 24.0° aP'.:!.. ~4.5°C. (Table XI). In the latter graph the mean of the logarithms of the times to death has been calculated. The very close approximation ot the median and mean mortality times is apparent and in agreemet't with the findings of Fry eta!., 194G. The median times to death have bef:'n plotted further in Figure 4a, illustrating the levelling-off of the median resistance times at lower temperatures to become parallel \vith the time axis. In Figure 4b the resolution of these data into a linear series of points has been achieved. The line A-B drawn almost at right angles to that for the median resistance times for 5°, 10° and l5°C. acclimations denotes the po;nts of time at which continuation of the experiment provides no change in results (up to 1-0,000 minutes). Although the resistance times of the higher acclimated fish (20° and 24°C.) showed an increased t0lerance at comparable times for periods of 1,000 minutes (about 17 hours) and less, continued exposure resulted in continued mottality to a level of death (line B-C) which fi.nallv equalled that of the lower acclimation of l5°C. This phenom~non has been ob- served repeatedly over the higher levels of acclimation fc.r every species studied by Fry et al., (1942, 1946) and Hart (Hl-4:7, 19-!9). It is a characteristic, to a greater or lesser extent, of all the species considered in the following presentation. PINK SAL"!\!ON. The pink salmon were decidedly the most diffirult of the five species to handle in fresh water after the first month of feeding, and for their length showed the least \Vcight (Table III). The difficulties experienred in arclimating this species to 24°C. were noted em·li~r. Their intolerance to a tem- perature as high as 24.0°C. is also apparent in the distribution of their resistance 1Complete tables of data are included in a Ph.D. thesis, 1951, in the libraries of the Depart- ment of Zoology, University of Toronto, and the Pacific Biological Station, Xanaimo, B.C. l ~~ l I : ( ' I : I ' l· I I t\ ~ ·'I I ' l ~' \ 't' ~ 1 ''1 : L r ! : .... ,. .. ' :~ l '" r ! ! I l . I I . I I I .. 1. \ l I ,. : 1\.f . . . - . . 278 ,-, ;· 7 • I 90 r-. • • • 26·0° I I 25·5° j 25•0° .. y. \." .. / j/ I I 70 • • ;· • I I I ~ • • 0 I I I· < "' 0 50 • r • -50 "lo Morfali!y I /· .J ... • ( • z I I w 0 a:: w • • • ;· n. I I I • • • fl I J /. I 10 /:!. /~ • I / I _j 0 200 400 1,000 TIME TO DEATH MINUTES FIGURE 3a: Times to death at different lethaltest temperatures amdng young spring salmon acclimated to 10°C. Plotted on arithmetric axes. l 90 I • I I • 255"1 ~ I I 0 "' tJ w 0 ... z ~! j w () a:: w • Q. ) u j I \ . ~ .~ . I ~ . ' 1. 260'1 70 • I 50 I I 30 10. i ;: "' I 10 100 TIME { t ro DEATH • Minutu X: Geometric mean tesistance time. FIGURE 3b. Times to death at different lethaltest temperatures among young spring salmon acclimated to l0°C, Plotted on probit X logarithmic axes, Calculated geometric mean resistance times coincide with the· median resistance times (at probit 5.0) • f :~ r-~------ . _ ... rr~~n1Uillu:rlJ•••~ iW &44 • *""" ' ,, . 1.) . "' a:: 26 ::;) ,.. c a:: w 11. 2 w ,.. 24 2 FIGURE 28 I I I' l t,) i • I· t.l 26. a:: ::;) ... c a:: t.l II. :! w 24 ... .. ~ FtGURE 4 acclimated ~ I I f j I t ~. I I' f' . r -~ . . 278 0 <( U.l 0 .... z U.l 0 u:. 1&.1 II. 0 < 11..1 0 ·-z 11..1 0 a: 1.1.1 0.. 90 70 !50 30 10 90 70 50 30 10. l 7 ;· 7 I • • • • 26·0· I I 255· /25·0° 24·0 \... ../ j/ I I • r.; r • I I I • Cl • • I I I· I • • • --50°/o Mortality I I. /· • • I I I • • ) ;· I I I • • • • I J /. I //. /. • / I 0 FIGURE 3a.• 10 200 400 6()0 l;JOO TIME TO DEATH MINUTES Times to death at different lethaltest temperatures among young spring salmon acclimated to lV°C. Plotted on arithmetric axes. I I • • no·.f 25·5· I • .I • I I i • f I • " I .. • /· • • /~J IQO TIME T'O I • 25{)"' • I • I • I • 1 • I • I '·· I DEATH .I .. ..l I • 1 ;: • I • I • I LJ. t,ooo M :nutes X: Geometric m•an reshlance lima. Fr:tURE 3b. Times to death ut different lethaltest temperatures among young spring salmon acclimated to 10°C, Plotted on probit X logarithmic axes. Calculated geometric tnean resistance tknes coincide with the·. .,.;.b.n resistan('e times (at pro bit 5.0). r '''c-~-.::-:~-.::: ~~JIIo ... T. :· 1 1 • 24 22 t. FIGURE ~b. acclimated l I ! l l I, I I l ' ':L 'I ; ·i .. ' '! ; 1~ f I t !/! -1 '; !l i ,, l \.~ ~ ., ~ t; 11 l.; ~ : ;j l· I I• . ;; ::~ 2 ;·. ' f l . ~,/'· 'l 1 ·-~. ! ·~ r [ rl f;;. • II J r > ., r ~~ f ~ f t J i ~ "" . ' ...,. . . . . ·:. ' .. ; .'" . . -. . . . . ...... . !- ~ 28 oe \\ (,) . , ~·\. Acclimaticn temp. w a: ;:) 1- < a: w IL !E , ... 1- ~~""'· 24° \\ ~ 261-0• 0~·--~ \. -------0 ---~-- 1 \ " -o/15~ --h> 0 ~ I \ •o• • 0 .,/' I ~--------------·--------------------24 • \ • • \ • 50 \ ~ • 22 !- II I I I I I 0 2 4 6 8 10 T I M E TO 5 0._,• M 0 R TALl T Y 1,000 Minuh;, FIGU...,.& 4a. Median resistance times to high temperatures among young spring ::.almon acciimated to temperatures indicated. Plotted on arithmetric axes. 2'8- ..; • 2 2. -----·----------------------------------------~ Acclimation TtmiJ. z••......._ it ~ ·~ 2 o• ·~ 10".......... -...........__. • <>-~-9 ~ ~.~ . ~.......... • 9 •• 5" "-.. ~ ""' '()-~ ,e_------~---•,.E •" .o-I ·~ ,;---------.----- • I "v~----------------A TIME TO 50% MORTALlTY -Mlnultl 279 FIGURE 4b. Median resistance times to high temperatures among young spring salmon acclimated to temperatures indicated. Plotted on arithmetric X logarithmic axes. (See text for further explanation.) - IIIII A •: I ~ . . l "",r .. ~ . ~~:...:.:~_:._-· · ---·· ·iiru:+Ce~ ....... I l I ,, r 280 times following a minimum period of acclimation to this level of temperature (Table IV). After 2,000 minutes' exposure to 25.5°, 25.0° and 2-1.5°C., no increase in their tolerance over that for 20°C. acclimated s<?mples was obtained. The results for lower acclimations (Figure 5) were in accordance with general expectations except for the 5°C. group. ~Iortality was so rapid in leth;:tl tanks with tempeqt.tures of 22.5° and 23.0°C. that it was decided to test the majority of the remainder of a limited sample at 21.0° and 19.0°C. Only four fish finally u . L I ILl 26- a: ::> 1- c( ' a: r-- w I ~ I w 24 -· .... I I_ 22- Acctlmatio" Temp. TIME TO 50"1'• MORTALITY -Minutes FIGT:RE 5. :\Iedian resistance times to high,temperatures among young pink: salmon acclimated to temperatures indicated. The encircled point for 5°C, acclimated p:inks denotes the use of four fish instead of the normal ten in other points. remained for an intermediate test at 22.0°C. The reliability of these cbta is therefor~ not as great as that for the more orderly points of higher acclimations. socKEYE .?AL~roN. Sockeye salmon, although held at a maximum acclimtttion of 23°C. (thus 1 °C', lower than the highest for pink salmon), also showed an inherent intolerance for such levels of temperature as 2-1.0°C. and 2-1.5°C. after prolonged exposure (Table IV, Figure()). Howev~, of the five species, the sockeye are probably the best adapted to fresh water, often spending two years in lakes before migrating seaward. A completely fresh-water subspecies is also commonly reported (Oythond, 1030). The responses of the young sockeye to high ternpera- tures were quite similar in pattern to those of a fresh-water species of Salmonidac. SalveUnus fontinalis (F'ry et al., 19-10). The lines connecting median resistanrc times for acclimations of 20°C. and below are fairly regularly spaced and are not significantly different in slope to be <li~tinguished as not parallel (Table X). ,, 1 i \ t \ I I \ I mately u . w. a: ::> ... <C a: ILl Ei ... , "" I CHC, in. Table for expe 1,000 mi in life al Chit1 an after 9,0 half the · to 2-1: °C .. COif_' the stoc ted som ...i;. I l ·I I I· I l l l tu[i ease • I I . ' t 281 The apparent variation in slope for the l5°C. acclimated group is not beyond what might be expected from chance. This has been derived from a consideration of the total data mustered in the tables of analysis of variance (Tables VI to X). As a result,.in this instance only, the line for the l5°C. acclimation has not been drawn by inspection as the best straight line for the plotted points, but as the most likely relation to be expected on the basis of the total data, that is, approxi- mately parallel. t A'<limolioo Torno. 281- 1 l L ' I W2GI-- c:: I ::l : 1- "' l 0:: .:-- UJ 1 Q. i ~ 241- 1-I ~ 22L I to• s• -o-I "'-. I ;t----------·1 .I I -------------1 lt,.... _ ____;t.....__...J_...L-LI _t_l _t_! ..LI url __ .t__.._j_J_I I I l I I l i Ill! 10 100 1,000 10,000 TIME TO 50"1~ MORTALITY -Minutes FtGt:RE 6. ).ledian resistance times to high temperatures among young sockeye saimon acclimated to temperatures indicated. A somewhat lowered re::.i::.tance among the 23° and 20°C. acclimated groups for prolonged exposure to 24.5°C., below the expected level, is indicated by the dotted line on the extreme right-hand side. CHUM SAL1ION. The results of experiments with chum salmon are recorded in Table VII and Figure 9. This species showed the greatest amount of variability for experiments in which the mean resistance time approached or exceeded 1,000 minutes. Like the pink salmon, the chum normally move to sea quite early in life although some have been maintained in fresh water up to two years by Chirt and Kuroda (1935). Acclimation to 23°C. was quite successful. However, after 9,000 minutes' (6 days') exposure to 2-1:°C. in a temperature-tolerance test, half the sample had died, ~onfirrning the impossibility of aedimating this species ·to 24°C. COHO SA.L~[ON. An accident in the early history of the coho salmon eliminated the stock of eggs held in one of two troughs. This loss, while unselective 1 necessita~ ted some curtailment in the programme of study. At the time of experimentation 4 ,hOt ... ,... I. ~ . .I I I I l I I I I l l l' I ' l r ' . 282 28 22 - 10 FIGURE 7. Acclimcticn Tamp. I I II !Ill 100 1,000 10,000 TIME TO 50"h MORTAL!TY -Minuln Median resistance times to high temperatures among young chum salmon acclimated to temperatures indicated. Acclimation Temp. I u..I __ _,____,_--1-_,LJ I I I I 10 100 TIME TO !10"1• MORTALITY -Mlnutu FIGCRE 8. ~ledian resistance times to high temperatures among young coho salmon acclimated to temperatures indicated. --- I l 1 f • 1, l ~ i • I t J I with upper} coho were . the spring f. permit morl ' In retrosp between c temperatu. Ther PROLO high temp 5°C. Thes ~ exposure \VI. In this res1 sockeye re l salmon, it· conditions~~ tures have~{ one week)\ The results' Figures 9 al 3,000 minu that for th first day (t period a rj chum salm · dir~ction oj penmentat1 LOWER LL SPRING· lethal level species amt with 50 ped total of 20 · times at 5 of removin. checking'' a single tes . mortality. j No siz~ on heat-told ever, were ;1 such that tl sample was was on han. )AU I t r I l \ i " ( I l l I \ I ! I I I l l l I I I I l l l l I ' l 1 l I ! L r l I l I l I f F L [ ~~ ~f b • 1 f ) i f • • I l l ··.1\ ! . l 283 with upper lethal temperatures, ic was apparent that at higher acclimations the coho were conforming very closely to the reaction times already determined for the spring salmon. The remaining tests in this series were therefore bypassed to permit more intensive investigation in the relatively unexplored low-lethal range. In retrospect this was perhaps unfortunate as it now appears that a difference between coho and spring salmon, where such occurs, is present in their high- temperature tolerance from low acclimations. The results for the coho appear in Table IV and Figure 8 . PROLONGED EXPOSURE. An experiment concerning long-term exposure to high temperatures was performed with sockeye and chum salmon acclimate(l to 5°C. These fish do not feed readily at critical high temperatures, so prolonged exposure would inevitably cause death from malnutrition. if from no other cause. In this respect the two species are alike. Since they differ in mig1atory habits, the sockeye remaining in fresh water usually for at least one year longer than chum salmon~ it is quite possible that they d11ier in ability to tolerate fresh-water conditions (cf. Hoar and Bell, 1950). So far, few experiments with high tempera- tures have involved exposure times in excess of 10,000 minutes (approximately one week), particularly for fish from acclimation temperatures below l5°C. The results for 30,000 minutes' exposure for these two species are recorded in Figures 9 and 10. The sockeye typically showed no additional mortalities beyond 3,000 minutes. The pattern of mortality in the chum salmon was very similar to that for the sockeye, with no further deaths. recorded between the end of the first day (1,440 minutes) and the end of a week (10,080 minutes). Beyond this period a rather unexpected but orderly progress of mortality appeared in the chum salmon samples. Speculation as to the cause of death might be made in the direction of the relative intolerance of this species to fresh water; rurther ex- perimentation is desirable. LowER LIMITS OF TEMPERATURE ToLERANCE SPRING SAL:MON. The method of determining the resistance times at low- lethal levels of temperature usually involved two samples of ten fish of the same species and acclimation for each test temperature. The removal of one sample with 50 per cent predicted dead changed each test :Jefore completion from a total of 20 to a total of 10 subjects, hence the plotting of the individual resistance times at 5 per cent intervals changed to 10 per cent intervals follo\ving the time of removing the first sample. The slight inconvenience involved \Vas a result of checking "predicted" against "actual" dead. In this manner all mortalities from a single test temperature could be plotted in determining the time to 50 per cent mortality. No size effect in Pacific salmon of the same age was demonstrated from data. on heat-tolerance (Table XIII). Results of experiments on cold-tolerance, how- ever, were strongly suggestive of a size influence. The method employed was not such that the individual fish, once dead, could be later identified when the whole sample was removed from a lethal bath. A group of fish, some living, some dead, 'Was on hand. By considering only those rases in which at least three hut not &StAt . . , ~ , ~: . '"'P • , , ~ . . : , . " . "' .. ' »'" --," ' -\ -.• ?· .. ,: ... . . . . ... ' ~ ~ . . -..... I l 284 l I 1 more thanA showed ne /" I sample as l I 90 0 . /--------------j I weights we, t l I were: livin, I • l statisticall • j I • \ 0 70 Evide' ~ ·I ;· w ( the first, o 0 • 50 and was fo • 0 I 1-24·0° I 23·o·f the metho 2: • w I -------------remainder.· 0 30 • 0 n a: l j { recorded o, w ~ a.. size factor~. I . / • firmatm;:. ~ 10 r 0 comparat1 l I I 21·0° None die stitute the{ -------------l Prelil" I ! had been i f I 100 1,000 10,000 2-:1:0 and 2 \ I TIME TO 0 EATH -M inutea ti with upperl I FrGt:RE 9. Times to death at various high temperatures among young sockeye salmon acclimated to 5°C. a3d tested for 30,000 minutes (approximately three weeks). I l l 90 /. I tempe I 0. 0 ;· I of death. I I !'' 0 • 0 ' 70 I l 0 • 0 l I c( I l w 0 0 L !50 • i I 1-··l l • 0 2t·o·J standing z 22/ I l w 23·0· 0 30 • 0 stopped. a: j· I w • 10. I 10 I l -! I I temperatu Even 10c-( I (three day 100 1,000 10,000 those ta I The I TIME TO 0 EATH -Mloutu I nounced FrGrRE 10. Times to death at various high temperatures among young chum salmon low tern l acclimated to 5°C. and tested for 30,000 minutes (approximately three weeks). I I l l l r L: I j'l f 1 i'· l ·~~ ·. i f 4 I , l i, ,. t ' l'. . ·::t ,, . .I 285 more than seven had succumbed, comparison of the sizes among all specimens sho,ved nearly twice as many living fish above the mean length for the total sample as dead fish. Since the measurements were made some time after death, weights were pot taken. The mean lengths and standard errors of the two groups were: living = 5.00 ± 0.046 em., dead = 4.75 ± 0.043 em. The data show a statistically significant difference (Table XIV). Evidence was procured for t'~.VO separate lethal effects of low temperature: the first, occurring only at the lowest temperatures, was very rapid and decisive, and was follmved by a second series of deaths after considerable delay. Owing to the methods employed, the first mortalities could not be sorted out from the remainder. They constituted a relatively small fraction of the total dead. Fn- recorded observation at the time of an experiment revealed an apparently distinct size factor in this primary phase of death, the smallest fish dying first. Con- firmatory experiments will be required for adequate proof. The presence of t,hese comparatively smaller fish in the samples of dead fish recorded above may con- stitute the main source of difference between the two major group~. Preliminary tests with spring salmon, before the system of predicting death had been instituted, were performed at 0.5°C. intervals from 0° to 3°C. (Figure 11, 24° and 20°C. acclimations; Table V.) The reasoning, derived from experience with upper lethal experiments, was fa.IIacious in part since the range of low tem- peratures causing death for 24°C. acclimated salmon was almost twice as great, within the same t!.~·ne limits (covering 6 rather than 3 degrees C.). The doubly "expensive" technique of closely spaced temperature tests coupled with sampling for dead t 1 ·roughout the experiment was replaced by the predicting system for tests at 1 °C. interval in the remaining cases reported for the spring salmon. The median resistance times follow a rather varied relation '\vit!l increasing temperature (Figure 11), and are apparently complicated by more than one cause of death. The sigmoid shape of the curves is one characteristic which persists throughout the remaining species, particularly at higher acclimations. PINK SAL~ION. Although a few preliminary tests were made with l0°C. ac- climated pink salmon, 5}/2 months old, mortality in the fresh-water holding troughs appeared during the last two weeks in sufficient proportions to signify some intolerance to these conditions. 3mall samples were incapable of y.,·ith- standing 5°C, Attempts to continue work with this species in fresh water w-ere stopped. SOCKEYE SAL1ION. The inability of Pacific salmon to tolerate sudden im- mersion in· low temperatures was well exemplified in the work with young sockeye salmon. For survival in nature the necessity for this species to acclimate to low temperatures is emphasized by the results illustrated in Figure 12 (Table V). Even l0°C. acclimated samples succumhed to 2° and 3°C. within 4,000 minutes' (three days') exposure, and~ temperature of 0°C. caused some mortality amongst those taken from holding tanks at 5°C. The sigmoid pattern of the resistance time-temperature curves is most pro- nounced among the sockeye. An initial period of rapidly increasing resistance to low temperature (for 20° to l0°C. acclimation) is followed by relatively little ,_ . . '. i '· . I . : ; ' I ! l ,. ( ' l I ! ! ! I I I I I I ' l 1 I l I l l l I I I l l I • I i I I ) I l '•. , ,. . • . . " I \~ . • --. ' • . •· . . , .-• .. , "~ II_ \ • • •• • • -.... ~ \" • • -. . !. "'"' -. .. . .. 286 (.) . 0 Acclimation '1!:, '.I . ~. -- ' -·-· ~-....,......,..._. ~ ~\ . 2 3 4 TIME TO 50~. MORTALITY -1,000 Minutu 5 f I I 9 t FIGURE 11. l\tfedian.resistance times to low temperatures among young spring salmon acclimated to temperatures indicated. Arrows signify tests at temperatures which caused less than 50 per cent death for 5,500 minutes• exposure if placed above the line, or greater than 50 per cent by the time indicated if below the line. B Acclimation t T!!mp. 6 u • I I&J a: ::I f- <C 4 IY. uJ Q. ~ I1J 1- ---------::1 t I 0 • ~ I _.~0 0 2 3 4 5 TIMe TO sc•;. MORT ALIT'/-1,000 Minutes FIGURE 12. Median resistance times to low temperatures among young sockeye ~mlmon acclimated to temperatures indicated. Arrows used as stated in Figure 11. - tt'ttl 1 '1_. I l \ ,, I t L I f I l • l 1 l l r I ' l 1· ~ ' 1 l l I I 1 1 soc ting I clearly l °C. ca turcs); I 4:0 to G mortali I mortali caused l of layed l f .. . f " r l ; , .• I 1.: J L; !. m, 287 change in resistance for two and three degrees' increase in temperature. No mortality occurs within a degree or so above this latter zone even for a considerably increased exposure time. The presence of this "break" at an exposure time of at least 4,000 minutes for fish acclimated to l0°C. and higher (usually occurring at test temperatures of 2°C. and above) supports the overall experimental time of 5,500 minutes for lower lethal-temperature determinations. CH1Thf. SALMON. The greatest difference in response to low temperatures was e.xhibited by the chum salmon from different acclimations (Table V, Figure 13). \Vhen taken from 23°C. and put at 7°C., 50 per cent mortality occurred by 4,000 minutes. From l0°C., however, mortality at 0°C. was observed only toward the very end of the imposed test time. This was somewhat surprising in view of the scant but indicative findings on the intolerance discovered for the pink salmon, t0. which the chums appear to be most closely related (Milne, 1948). COHO SALMON. The data and curves m~·strating the median resistance times to low temperatures for coho salmon appear in Table V and Figure 14. The sig- nificant mortality among members of this species at 0°C. when acclimated to as low as 5°C. demonstrates how confined the coho are to temperatures above the freezing point of water. In general their reactions show the same trends as in the other Pacific salmon. MIXED LETHAL EFFECTS. Brief rr.ention was made of two distinct responses observed while studying the lethal effects of low temperatures. The presence of very rapid mortalities in CO'ltr~st with delayed lethal effects, either between. tiamples at slightly different temperatures, or within samples at given critical temperatures, was apparent from even superficial examination. Doudoroff (1942) observed somewhat similar phenomena among young greenfish, Girella nigricans, and distinguished between "primary chill-coma" and "secondary c\ill-coma". He records that "The initial shock was not manifest until several seconds after transfer to the low temperature, and apparently was not due to stimulation of the cutaneous sense organs, but was produced only when the low temperature had penetrated internally, probably to the central nervous system. Accordingly, it was more delayed in large specimens than in small ones". The discovery of a satisfactory criterion of cold-death :J.mong the Pacific salmon permitted a more critical study of the time-temperature-acclimation relations for death. Results for sockeye salmon will be presented, being more extensive but not unique. By plot- ting the data on probability X logarithmic paper it was possible to discriminate clearly between the two trerl~;:: of death. From 23°C. acclimation (Figure 15a), 1 °C. caused rapid death for all individuals (as presumably would lower tempera- tures); 2° to 3°C. spHt th€ samples into rapid anci delayed deaths as shown; 4° to 6°C. resulted in only delayed deaths; above 6°C., less than 50 per cent mortality was obtained. From 20°C. acclimation (Figure 15b) 0°C. caused rapid mortality; 0.5°C. divided the sample into the two types of death; l°C. and above caused delayed to no mortality. From l5°C. acclimation (Figure 1.5c) one case of mortality was observed for a lethal-test temperature of 0°C. followed by de- layed to no mortality at higher temperatures. The time to 50 per cent. mortality is consequently affected by per cent occurrence of ''primary" cold-deaths within the sample and by the size of the fish which chance to be present in that sample. ' I - ... .tt :. J ·j ' ·~ J " t t l'; . ~· ~ . ' i L I .. .. ' I ~ ; 1 ' I I ' w: !J Jl l I I I ' '-, I l I l L r l I I r \ ' I· I i ! I I l I i r \, l ! l \ 1 l L r \ l r I I I I \ I' i. I l I l l l t 'J"' r l h L L 288 tJ • «: I.IJ 1- Acclimation 0 • I t I t I t .~ 2 3 4 5 TIME TO 50c/• MORTALITY-1,000 Minutes FIGCRE 13. :Median resistance times to low temperatures among young chum salmon acclimated to temperatures indicated, Arrows used as stated in Figure 11. u • 4 Acclimation Temp. "" t ----·---------------' l -----~ t 10~-----, .~~~---~~~---~:-.~ l 2 3 4 TIME TO 50% MORTALITY -1,000 Minutu FIGCRE U. :\Iedian r~sistance times to low temperatures among young coho salmon acclimated to temperatures indicated. Arrows used as stated in Figure 11. ···--~,.-... l ...... Ci "' ( I.IJ 0 I 1-z I.IJ t u a: I.IJ Q. t. 10 Frat:RE ... 15a. 90 0 70 '"' I.IJ 0 ~0 1-z I.IJ u 30 a: I.IJ Q. 10 J • 10 FtOl;RE 15b. J.:,_:.; I I I 1 1' I l . l I I I 1 L 1 l ' r ~<. -. . . • • . '-• • ·, •. • • • • • • :. . • • ~ •• I ·~ #.• . .. • -• 1- 'it' .. < .~ [ . r ,. I· ~ L r.· f: -~ f:! i i: !: r· r r ~ iC_ Il rl II t I ~ I t I l L l t t ( I I \ 4 ~ I I ( ' 90 0 70 ct L1J 0 ... z L1J 50 u 30 10 I I I! 10 100 T I 111 E TCf 289 I I 17 c I We we. /'/ . • 0 ---- .. -.... ...... - 0 .J I I I I " 1,000 10,000 DEATH Minutes FIGUREr l5a. Times to death at various low temperatures a-nang young sockeye salmon acclima- ted to 23°C. Plotted on probit X logarithmic axes. 90 I .I I we. • I 70 I .. o·o··::. ···y • c ../ . ~ e'/ w • c I I • 50 • • ... l z •• • ;· • ~ 30 a: • w , 11. 10 10 tOO 1,000 10,000 TIME TO OE ATB Mln1.1tu Ftol;RE 15b. Times to death at various low temperatures among young sockeye'salmon ncclima- ted to 20°C. Plotted on probit X logarithmic axes. I. ,_"'-~··--•,r-··-~ : ...... -.. -......... _ .. ,.-- "~ ,, , , • ' .&iUfjljglflj@!liL;ti-~t '*' r ...... - I ~ ~~ l l ~ I ~ r ,I ~ l ' ~ l t l w ! l I I j ! I . 'i .. · ' .. i . I "'· ' ~ I I l i ; I , . .. I . l ! ! l. \ I 1 .. r 290 90 0 70 < .... 0 !50 ... z .... () 30 0:: .... a. 10 L / / o.o'c/ IJ \. 0 r·o·cf • \.!: /;•: we • or/ I . . 1/ r t r i I I II I 10 100 1,000 10,000 TIME TO DEATH -MintJfea FIGURE l5c. Times to death at various low temperatures among young sockeye salmon acclima· ted to l5°C. Plotted on probit X logarithrroic axes. A reason for greater variability in cold-temperature resistance and the variety of inflections making up ::he median resistance time-temperature curves can be attributed partly to these causes. The work of Doudoroff (1942, 1945) was suggestive of an osmoregulatory problem in the delayecl cold-deaths, possibly acting as an accessory lethal factor. To test the hypothe-~;1s, a preliminary experiment using two groups of 20°C· acclimated sockeye, one in fresh water and the other in Atlantic sea water diluted to 9.9 parts per thousand, was performed at comparable low temperatures (Figure 16). Both rapid and delayed ~old-deaths were observed at 0.2°C without significant differ-ence in response within the two media. At 0. 7°C. an indication of greater resistance amon~ the last surviving members in the saline solution was present. At 3.2°C. a decided increase in the resistance was observed but not to the point of eliminating death from low temperature in part of the sample. Two conclusions can be drawn from this experiment. At the lowest lethal levels of temperature a medium of salt water (slightly hypertonic) does not alter the course of death from that observed in fresh water. Such a medium does, however, reduce the lethal effects of low temperature for delayed cold-deat~ when resistance times exceed 1,001) minutes (about 17 31ours). From these tt would seem that three causes for death are involved: one, a very rapid agent usually effective within 60 minutes of exposure, a second, not so rapid in action, and a third which is re!ated to osmotic balance. -· f f t ( l • t t • l ' H I 'i . f. ,,___.._;.;._~.....:...._c..:::........-~~0 l 90 0 70 -< .... 0 !10 1- z: ~ 30 () a:: "" (L 10 ° . I FIGURE lJ acclimatedl (slig.ltly h The osmotics and Hoa ~\tlantic These fin lethal ten agent at from hig · involved SPRI.- of preferr lined und surfaces, demonstr 0 of the ide 0 I I 1 I l I I ' I l l I r r rfl '• ~· t ft ' !' 1 t l ( I l I ' I I ( . ' 291 0-Frtsh Wahr ~0 e-Sea Wattr 9·S%. I/~ 0 70 <l Ill • 0 Q. o·2·c."' :so 0 I ,--- 1-/Q// I :z 0 8/ w I/~ I 0 30 a: Ill c. 10 /~ o1,-~l /3·2·~/ 10 TIME TO PEATH Minutea FIGURE 16. Times to death at various iow temperatures for samples of young sockeye salmon acclimated to 20°C. and tested in fresh water and in sea water diluted to 9.9 parts per thousand (slightly hypertonic). Where a difference in :~sponse was apparent a broken line has been used for the salt-water data. The observation that size appears to have some bearing on resistance to osmotic stress was reported by \Vilder (1944) for sea-nm speckled trout. Huntsman and Hoar (1940) using salinities of 20 and 28 parts per thousand concluded that Atlantic salmon parr "as they increase in size become more resistant to sea water". These findings in conjunction with the size effect among Pacific salmon at low lethal temperatures support the possibility of an osmotic factor acting as a lethal agent at low temperatures. Conversely the lack of any size relation in deaths . from high temperatures might be taken to indicate other than osmotic factors involved in these mortalities. PREFERRED TEMPERATURES Sl.'RING SALMON. Some of the inherent problems concerning the determination of preferred temperatures for fish in a vertical temperature gradient were out- lit-led under 41 lVIethods''. The presence of other gradients (gravity, distance from · surfaces, etc.), if impossible to remove or control, must be ac<..ounted for by demonstrating the supression of these irt relation to preference for some level <>f the identity under investigation. By changing the position of the temperature gradient within the tank, without altering other relations, it was oossible to . . demonstrate the selective aggregation of spring salmon in the r.egion of 12° to ' \ I' l I ! i l l l I '] . :: ' ',, .l ,, l L ' ~ ., L t; J ,j 292 13°C. (acclimation of 20°C.) despite a highly significant difference in their position within the tank. This distribution in space, varied yet remaining relatively constant with respect to temperature, has been depicted for this species in Figure 17. An att- empt has been made to conform to the relative dimensions of the tank and distribut- ion of the fish, such that the figure presents a "graphical picture's in every sense of the word. z 0 U) > c X ao 60 40 20 o 20 40 60 ao FREQUf;NCY FIGURE 17. Frequency distribution of young spring salmon acclima- ted to 20°C. in three successive temperature gradients, using the same tank. The calculated preferred temperatures are noted for each distribution. i t I -I l t f I A few observations which apply to all the species are of significance. -:\o matter what the gradient, even reaching the lethal level, the fish wo.uld make feeding excursions to the surface if the appropriate movements of the investigator~ which always accompanied feeding, were made. On some, although infrequ<.>nt, occasions, under the routine procedure of !ow illumination and night-time rc-. J cording, no selected temperatures could be determined. Activity of the fish was too great, being more limited by the surfaces of the tank than by any other rc· cognizable feature. No suggestion to account for the increased "excitability" on these particular occasions can be advanced. Adequate feeding and unifonr·r quiet conditions were always observed prior to introducing a temperatun .. gradient. A natural "expJoring'' activity is a characteristic of the group. - peratt for ac : ~peciesi tole~arf spCC'IC obscrv' D -o,~ i L.Jl \. • , genera. tion ta ! greatt•d what ~~., ot tina! a~ \, 'o~IP.\ and in po~cd: tl (:3 ~ignifie ·: (4 : fll(."<lian varianc differcn of the f basic co an· lima nmside Sp analysi. 1 Refer I l f t ,.~ rj ~I f l ... ., 293 PINK, SOCKEYE~ CE:t!"t AND COHO SALMON. The distribution in various tem- perature gradient~ as inmcated by the preferrc,; -:emperatures (mean and mode) for acclimations, mostly includiq:; 10°, 15° a11rl 20°C. in the Pacific salmon species, are presented in the gra.ph.s used to display the zones of temperature tolerance (Figures 20 to 2.:1:). The vr:.rl.ation in ;:esponse between the different species was not sufficient to warr~nt dealing with them separately. Some general observations can be made. Despite considerable difference in temperature-al:climation, amounting to li5°C., comparatively little difference in preferred temp:crature \Yas ob~erved t::.1>.periment1.!ly. On the average no greater difference than 3°C. (11 o to l-!°C.) ~·:a~ displayed between means, and the region of greatest preference lay in the 12° to l4°C. stratum. The pink salmon from 20°C. acclimation constituted the only case in ·vvhich a preferred temperature as high as 17.7°C. was observed. The general avoidance of temperatures above l5°C. for all species, in $lpite of acclima- tion to thib level .:1nd to 20° and 2-!°C., was very markeci. A tendency to show greater dispf;rsal in the fish from higher acclimations is suggested by the some- what larger standard deviations for these samples. Only a record of the mode was availab!e for 5°C. acclimated sockeye on final analysis. Unfortunately no more supporting data are on hand. C0:\1PAR!SON OF TEMPERATURE RESISTANCE RESISTANCE TIM~~ TO HIGH TE:~IPERA.TURES. The extent to which acclimation affects the resistance times to high temperatures ·within each species and the levels of toleran ..;e characteristic of each species have been p_resented graphically and in tabular hrm. If comparison is to be maJe a variety of questions may be posed: (1) HO\v do these species differ in their resistance times at differe!lt levels of a1.Tlimation? (2) If a difference exists, is it a matter of a differe1tce in s1ope (rate of change of resistance time with temperature), a difference in overall level of temperature resistance, or a difference in both slope and level? • (3) vVhat measure of difft:rence can be quoted and with what statistical significance? (J: Can the postulated straight-line relation bet\veen the logarithm of .\~e median t~sistance time and temperature be justified? · Each of t.hese que,tions can he answered by the method of analysis of variance (AIJpendix I) which permits a sorting out of the sources of specific difference from those resulting from sample variability coupled with interaction uf the factors of acclimation, lethal test temperature and resistance time. Three basic comparisons can be made involving two of the variates-species, lethals1 or acclimation.s2-in as many cases as exist in terms of the third. Thes}! are now con~idered: Species X lethals, for three levels of acclimation. The results from three analysis-of-va~iance tables (Table VI) demonstrate a highly significant differem·e 1 Refers to lethal test temperatures. ~ Refern to acclimatmn temperatures. 4A '"'h~ . ·' ; it 1 \ I l ! l r ! I ! I l f l I l I . . ~ ,. • . . . ~ .. ,~ ' -~ 0 I . . v , -·· . " ,, -- .J ... ~~·-~~~-~C\¢~••V·>H·'·1···· 294 between species (P < .01) which increases with lower levels of acclimation, that is, at a lower level of acclimation temperature for a comparable treatment of lethal temperature the difference is more distinct. This is the crucial test. Figures l8a and b illustrate this relation. A further analysis to determine 1vhich species are contributing to the difference is considered later. As might be exp._cted, the 0 • LIJ cC j ... ~ a: LIJ Q. :IE LLI ... 21 I I Ill 100 1,000 10,000 TIME TO 50% MORTALITY -Minutes FIGURE 18a, Comparative median resistance times to high temperatures among young Pacific salmon acclimated to 20°C. Lines have been drawn for the most and least tolerant species. u • 22 FlGURE 18b. • y-Piok -' -====-= "'--Chum -f I II II I 1,000 TIME TO 50% MORTALITY Mlnutu Comparat~~'c median resistance times to high tempera 1~ures among young Pacific salmon acclimated to 15°C, J '•-····~-~--··--;-·~--'"" -·~-···~--.~···---•• ·: •' .. ••···•····~·••oe····~•····-·""' •·•• "' .. o . ·i'1~MI18ii'i.Wilillullliiill~~ ... it:l. -- \ I ' i .• ! differences f arc also highl Species is a case in ,,.,. lethal levels i results in Ta crease at }0\\' as that for a lower levels If a lethal have little <'On trast, t that will best Letltals of-variance com pari sons. temperatures. in each of '{< spedcs, the S<l:, the others ( , .tpparent fron It is poss s pedes X let It icspvnse amo levels of tern lethality, red of the three fi, the interactio · 3.0, and is su rpecies X letlz A further may be attribf. , in the first an<j the total sum , extract each (Fisher and Y for single deg and can be c Such a search findings from·, made by use o <'<Hion of the (1) Nosi, exists betwce (2) Spri:~ ' from that of c - I \' j, l' \ I l I [ 1 l [ I l I I I l I l f:' l l L.rw· Jj I --al:n, 1ent of , i~'es ,·P ·. es •. , d, the I; r: f r c ~> '.; mt~"' ·r~:,r ..•. :~· .. · .. ( l I ~ l 1 v ( I J 295 differences for all species in their response to different lethal test temperatures are also highly significant. " Species X acclimat·ions, for four levels of lethal test temperature. This is a case in which the lumping of resistance times of three accJimations for single lethal levels is used to distinguish between species. It can be concluded from the results in Table VII that between species at each lethal level, and with some in- crease at lower levels, there is a consistent significant difference, yet not as great as that for a series of 1ethals at one acclimation. The increasing significance at lower levels of lethal temperature is inherent in the study of temperature effects. If a lethal temperature approaching that for boiling water were used, it would have little consequence what species or acclimation characterized the fish. In contrast, therefore, it is the temperatures which just cause distinct mortality that will best demonstrate specific and acclimation differences. Letlwls X acclimations, for different species. These five "4 X 3" analysis- of-variance tables (computed in Table VIII) complete the series of first-order comparisons. In essence they s!gnify what has already been stated, namely, that temperatures of both acclimation and iethal level have highly significant effects in f!ach of the species (former tables only applied to the group as a whole). One species, the sockeye (P < .05), does n0t show the same degree of significance as the others (P < .Oi). There is a greater variability in this species \v-hich is apparent from the diversity in the points plotted in Figure 6. It is possible to dmw up a table of second-order comparison, for example, species X lethals X acclimations. The v~ry fact of a difference in temperature- response among the species which resulted in the use of somewhat different lethal levels of temperature, to bridge the c.uses from non-lethality to fairly rapid lethality, reduces this table for like comparisons below a feasible size. A survey of the three first-order tables, however, reveals that the error term, signified by the i:nt~raction component in each table, lies mainly bet'..veen values of 1.0 and 3.0, and is sufficiently consistent to support the view that the interaction of species X lethals X acclimations would also be con.3istent throughout the relation. A further step involves assigning the particular amount of ·variation which may be attributed to each independent comparison (degrees of freedom). Thus, in the first analysis concerning species X lethals at .20°C. acclimation (Tnble VI), the to1 al sum of squares was 1249.02 with 29 degrees of freedom. It 1s possible to extract t:ach independent comparison by the use of appropriate multipliers (Fisher and Yates, 1948; see Appendix). Since these are then the sums of squares for single degrees of freedom they also equal the mean square or variance (s 2 ; and can be compared for significance directly with the "error" by an F test. Such a searching analysis has been carried out in this particular case only. The findings from it con~erning the species X lethals, and the statements already made by use of d1e more generalized analysis tables submitted, permit the appli- cation of the following conclusions to the whole problem (consult Table IX): (1) No significant difference in response to upper lethal levels of temperature exists between spring and coho salmon (acclir:tated to i0°C. and above). (2) Spring and coho salmon show a highly significant difference in response from that of either sockeyet pink or chum.' - .. -.::;:o .. .. L%2ZJt 1 I ! I· I I I 296 (3) Pink and chum salmon show a barely significant difference from each other, but not'from sockeye in either case. (4) There is a very highly significant "linear fitness" of the logarithmic re- lation for these data which accounts for 98 per cent of the variance; the balance can be attributed to curvature. Of the total 29 degrees of freedom, 20 were assigned to the "error" com- ponent (Table VI). This component is constituted of the variability of the organisms resulting in slight deviations from the postulated relation and their interaction (lethals X species), plus the variations due to experimental procedure which can never be entirely eliminated. By the same method of using appropriate multipliers, 20 separate components each representing one degree of freedom were obtained. A study of these p: <·vided no value \Yhich in itself was signi- ficantly different from that to be expected from the variability of the material. Consequently it can be concluded that: (5) The slopes of the lines relating rosistance time to temperature for the five species are not significantly different (acclimations 10° to 20°C.) and (6) The same relation can be applied to each species with equal confidence, differing only in the temperature level at which this relation exists. RESISTANCE TIMES TO LOW TE1iPERA.TURES. The constancy of relations in the upper temperature levels is in contrast to that for lower temperatures. ~o systematic testing of the latter data is possible from present knowledge of the subject. A graphical presentation of some of the responses among the young Pacific sumon taken from the same acclimation temperatures (Figure 19a and b) serves to illustrate this phenomenon. A fair similarity in pattern for 23°C. acclimated species becomes progr~gsively more variable with decreasing acclima- tion. The chum salmon 1 which at first seemed to be among the most sensitive judging from high acclimations, were the ieastsensitive from an acclimation of l0°C. The spring salmon were consistently the most sensitive to the lowest tem- peratures (0°C.), but showed a rapid increase in resistance at slightly higher temperatures. A striking intolerance to low 1emperatures characterizes all five species. Further study in this field of temperature-responses will be necessary to clarify some of the complexity which has appeared in these findings. The possible in- fluence of size has been pointed out. The mean fork-lengths of the fish used are reported in Table III. ZONES OF THERMAL TOLERANCE The· concept of a zone of thermal tolt>rance bounded by upper and lower incipient lethal temperatures for the greatest possible ra,nge of temperature- acclimation, and terminated by ultimate lethal temperatutes, was advanced by Fry et al. (1942) .for the goldfish. The freezing point of water limited the minimum acclimation to 0°C. for fresh-water fish. By construction of a trapezium relating these confining temperatures, calculation of the area of the zone of tolerance in "degrees Centigrade squared" gave quantitat~ve expression for an otherwise quaHtative description. Various species of fish have since been des.cribed in this , i ~ l • \ I l ~ I f FIGURE 19a. salmon 6 <.i • w a: ::1 .... o( 4 a: w a. 2 .., 1- 2 l J • f I 'lh uc re- f I t 4 I 4 f w a:: ;;) .... < a:: w c. :::!! t:r .... 297 --::=:==========:..-:-:-::. Sprino -• Chum - o 0 t • ----------------Sockeye-& ' ~ Coho -~ • 0 ;------·- .Vi"J ·o n· ott____.o ______._--.....1--_.__--.J-..-_____. 0 2 3 4 TI':4E TO 50"/e MORTALITY-1,000 Minutes FrGURE 19a. Comparative median resistance times to low temperatures among young Pacific salmon acclimated to 23°C. Arrows used as stated in Figure 11. + " 6 0 ..... a:: ;;) 1- < 4 0: w Q, ~Chum -o t Sockeye·& ~ ·-··················· ••••••• Coho -~ w .... Sprino -• 2 0 TIME TO 50% MCRTALITY-1,000 Minutes FtGt ~ 19b. Comparative median resistance times to low temperatures among young Pacific salmon acclimated to l5°C, Arrows used as stated in Figure 11. ' I ·-···---··---.. -------. --~ '1 :.; • \ -~---'"~--~~~· ~~- \ l I I I I ! \ l I I I L r- \ l ! l l t I i \ I ! I I I I l !' ·' 298 fashion (Brett, 1944; Fry et al., 1946; Hart, 1947, 1949), all conforming within slight variations to the original pattern for the goldfish. An increasing acclimation temperature has always resulted in an increasing lethal temperature in some fractional proportion. The construction of a diagonal iine at an angle of 45° to the axes (Figure 20) has provided a ready means of determining the ultimate upper iethal temperature, since this line represents the locus of all points for which lethal temperature equals acclimation temperature. CharacteristicalJy, for upper incipient lethal temperatures, no spedes has been found to exploit the , full possibility suggested .bY e.xtrapolation of th1s linear relation (between upper lethal temperature and acclimation temperature), always dropping short as a result of uniform intolerance at the highest acclimations, providing a "plateau" in a graphical presentation. Although the resistance time to a high temperature may be lengthened through higher acclimation, it was pointed out that this re~ sistance is finite and the lethal temperature remains unaltered for the relation designated by the "plateau';. These relations are illustrated iP '~igures 20 to 24 for the Pacific salmon. Some new aspects,· cliff ering from the nor 1t1al trapezium, are apparent. The lethal-temperature points are not always best represented by a straight line, particularly in the lower lethal bracket. A high degree of sensitivity to low temperatures among the Pacific salmon almost confines these species to acclimation temperatures above 0°C. Some death at this level was observed among 5°C. acclimated samples (Table XII). In a preliminary acclimation culture of spring salmon at aoc., high mortality occurred in the presence of healthy sockeye, chum and coho salmon of the same temperature histol\y. This intolerance of the springs might account for the rapid falling away of the upper lethal temperature for low acclimations, which is not apparent for the coho salmon, so similar in other respects. The spring and coho salmon had the greatest tolerance and were practically identical in area (529 and 528 degrees C. squared respectively). The sockeye were i~termediate (5()5 degrees C. squared) and the chum salmon least (468 degrees C. squared). The line relating upper lethal temperature to acclimation temperature for the pink salmon was very similar to th.at for the chum salmon Th~ apparent intolerance of the pinks to low· temperatures would restrict their zone of tolerance even more than in the case of the chums, placing them lowest in order of eury- theqnal relations. \Vith the possible exception of the pink salmon, and notwith- standing the variable nature of the lines relating lower lethal temperature to acclimation temperature, most of the difference in areas is a result of difference in upper lethal temperature. DISCUSSION AND CONCLUSIONS TIME AND TEMPERATURE Division of response to extremes of temperature into zones" of tolerance and resistance, previously set forth for other fishes, has been appropriate for similar distinctions among the Pacific salmon. Although the pattern of resistance to low temperatures was quite di(ferent from that for high temperatures, the same factors of tolerance and resistance were equally applicable. - I l f the .. ,. ....... '"' ....... These ha and Beleh "there are .· due to the Ft pr wi c \\" extremes of whit·h an o tern pcratu r · chnractcris in tlucnecd , · when the r s~tmple at which no m hns been e.1· tur. cs (h.igh .• .. · .. • <:an be dete . ' • I I I ~0 I \na. ·; for .. .. 'J.t,ure 'he~· , J I ,;l.il . ~ _. ··~ ·¥~ry-:fntl : ... r . ~fe .~ ence l I l 1 1 ~ () l -~.1..~.---...L 299 Heilbrunn (1943) has compiled a considerable number of records concerning the temperature at which thermal death occurs in a wide variety of organisms. These have been taken largely from reviews by Kanitz (1915), Uvarov (1931), and Belehradek (1935). In criticism of these data Heilbrunn (p. 420) comments "there are very fe·wr useful records of heat (or cold) death temperatures. This is clue to the fact that many authors have neglected the time factor". Death from w a: ~ ... 4 a: w Q. :E w ... 0 .t:! -.. ..1 ~ .. .. .. • -.., .. Q. 0 25 = 5 .. ..1 .. .. • 0 ..1 0 5 10 20 25 •c. ACCLIMATION TEMPERATURE FIGPRE 20. Thermal tolerance for young spring salmon in fresh water. The preferred temperatures are represented by a central point for the mean, with limits for one standard deviation dotted above and below. The degree Centigrade in which the mode occurred is represented by a s01id vertical line. Where an experiment was performed but resulted in less than 50 per cent mortality a "V" has been inserted. extremes of temperature is not just dependent on a threshold level below or above which an organism either lives or dies, but may be considered a resultant of both temperature clnd expo~ure time. At each lethal level of temperature there is a characteristic rate of dying (rate of mortification, Fry et al., 194G) which may be influenced within limits by acclimation. A threshold level is appi ;ached, however, when the rate of dying approaches zero, as in the case calculated for half the sample at the incipient le~hal temperature. Consideration of the time beyond which no more mortality may be expected from tempei·ature as a primary cause has been extended in the present study for low lethal temperatures. Tempera- tures (high or low) which would not cause mcrtality, regardless of acclimation, can be determined from the zone of tolerance. Very similar limits can also be set - ~· il f ~ l :li " ·~ ·ll !! 1: •' I: 1i 'j> ~ t: , I I i ~ l l I I 1 1 J I I l ( l • I l 1 I ! t ' l l l I ! l l I i I d • 1··.· I I 300 &~ -0 ~ .,.. .. ...1 ... ·---·----·-· ---·------... Ci. zo a. :;) Il 1 15-- "0 .. ... ... .. -Il .. ... 0.. i / 0 ACCLIMATION TEMPERATURE FIGURE 21. Thermal tolerance for young pink salmon. No !ower lethal experiments were performed. Preferred temperatures plotted as in Figure 20. ~c. 0 .s: -.; ...1 .------------;t··-1 --·-----... ---... .. ... 20 <>. ~ w a: :;) 15 yl! ! ... "0 "' .. 1~ a: ... . ... w .. . i -a. .. 2 ~ 10~ I 1.1.1 1-\ I , , •I 0 s: 5 ./ -.,.~·-.. ..J .-------_ .. .. ., L, . !; 0 ..J 0 5 10 15 20 25 ACCI.!MATICN TEMPERATURE FIGURE 22. Thermal tolerance for young sockeye salmon. temperatures plotted as in Figure 20. •c. Preferred ,. . ,)··.·~ ·.:]l .. ,• :~:; --... ~ a. 2' w 1- \ \ Ftm' I I t I r I' v• • ( f .. , l ;, I I ! L l l l \ J I I ·- I I w a: ::» ~ < a: o.J Q. :E IIi ... " J:: -o; ..1 "' • .. .. "' -• .. :g. " £ -• ..1 .. .. ~ 0 ..1 --- 15 10 5 0 __ o li:---··-----·--- T /It It l! ! ..t / .~ ~· + __J! s-ICi 15 20 ACCLIMATION TEMPI;RATURE 25 •c. FIGURE 23. Thermal tolerance for young chum salmon. Preferred t~m­ peratures and "V" plotted as in I<'igure 20. ILl a: ::» ... < a: ILl 0. :E ILl ... 0 .<:: -.. ..1 "0 .. .. .. .. -~ ~ 0.. ;; .c; ; ..1 .. .. ., 0 -' 5 I 5 10 15 20 25 •c. AC CLJMATION iEI.IPER ATUR E FIGURE 24. Thermal tolerance for yottng coho salmon. Preferred tem- f , perature plotted as in Figure 20. · :. i 'J ' ' '~ ., ,. ·.· ·.;J· ~' ,. ~.~', ., " ..... ~-. -·-··········-····~ ....... ., . ., .. -·-··· ·r .. t --1 . I 301 '· . ~· t r I l. l r· l l I ! l j l [ I I I .~,. ! i •• I ' ' ~~~ ' ' ~\ \ 302 by examining the resistance times for various acclimation temperatures as in Figilre 25 for sockeye ~~lmon. The points be:F.md which no more deaths are likely to occur have been h.dicated by a dotted line. Thus, temperatures between go and 21 ~c. are unliL 2ly to catwe death among sockeye no matter what the acclimai:ion, nor what the exposure time. The higher or lower the temperature is above 0r below these limits, the longer the test time necessary to determine the lethal temperature. A somewhat different series of ~ethal temperatures would have br-'.;fi quoted if no experiments had been continued beyond 24 hours (1,440 min,) for both upper and lower lethal levels. Future experhnents on temperature-tolerance will continue to add to our knO\vledge of the factors affecting temperature relations in fish. In the investiga- tion of Pacific salmon, emphasis has been placed on uniform treatment of ail samples such that, although the methods and later analyses may change, the differences set forth should remain unaltered. One limitation should be pointed out. The results apply to the stocks of salmon from which the eggs were collected (Table I) and may therefore be traced back to comparatively few females. The possibiJity of variation both within and between local stocks cannot be disregarded. COliPARISON \YITH SOlfE OTHER S.-\L~fONOIDS The resistance times for six species of salmonoids determined for samples acclimated to 20°C. have been plotted in Figure 26 (from Fry, 1947b; present paper). Data for only two of the species of Oncorhynchus are included. These two, the spring salmon (0. tschawytscha) and the chum salmon (0. keta) wtre res- pectively the most and least resistant to the same test temperatures; the re- maining three ·species occur at intermediate positions (Figure 18). It will be seen that the two species of Salmo occupy positions distinct both from each other and from the ~emaining species. Salmo salar, the Atlantic salmon, is the most resistant of the salmonoid group. The members of the Pacific salmon species lie in a compact series intermediate between Salmo and Cristivomer, while Salvelintts fontinalis approaches the resistance of Salmo trutta at the highest test temperatures (28.0° and 28.5°(',) but drops below it for lower temperatures, falling within the range for Oncorhynchus. Except for the speckled trout (Salve/in us fontinalis), no further comparisons are \Varranted until more experimental data are obtained. A marked difference in the lower lethal relations between the speckled trout and the Pacific salmon exists, sufficient to suggest a qualitative difference rather than a purely quantitative one. The former was found to be resistant to low temperatures by Fry et al., (104G) who report that the lower lethal temperature "was only just above 0°C. when the acclimation temperature was 2-eC''. This results in a comparatively large zone of tolerance (025 units: exceeding that for Pacific salmon which ranged from 408 to 520 units. SOME ECOLOGICAL RELATIONS Limits of tolerance to extremes of temperature among young Pacific salmon have been determined, Significant differences exist, \Vhether these differences are sufficient to account for some of the distinctive habits which characterize the different species is a matter of conjecture. 1 I i J r t -• i t \ 30 28 22 20 0 • 19 ""' a:: ;::) ._ <t a:: ""' c. :::e w 1- 16 14 12 10 6 4 Fmt:RE 25. Broken lin~s . .I ~. l \ \ t f l l i I l ! : I d r ...... . (::' "" . ' --. . . • L.:;. *~,,.. .. :.,, .. ~ , •••• '~ '' )-o'' ..:. r I he ll<J .tcw ;r~ al.- he ~o~~ D . 1 xes : ::1 ~~ ~~~ ch hf• '. ~ .1e ·He , :!St es~ .t'li. ' ~ta '·h~ ' ' ' ' ~ Vt ''be- 1e~ tn ts1 I ' on l I I \ \ ·I 1 \ ~ '( I 303 D oys 28 t\il; • Acclimation Temp. 26~, 12 '-:._--:_--:::,'Jil::•-~'.,___ ..................... -··-···-···-·------··-~-:--···-@). _ _g~~~:-- 24 .\~ --.. -~·----.---------------- \ ....___ . ..-· 10° \ @-,.·------------------- ·........__ ••.•. -·· 50 ..,.. ________________________ _ 22 ./" •.. ·· ......... 20 (.) 0 18 w a: :J 1- 16 <t 14 a: w a. ':E w 1- 12 10 Ac c lim ati on Tern p. 8 ·····-················· -.... ., 6 4 / ?···.-.. ------23° ··. ......... •... ... ... , I .. 2 0 21 ;· ·· .... \. ~ . ' 0 1___... I • ./-.o/, ' I \. _s_o-1---~--~---~'------~ I 2. 3 4 5 6 8 9 10 TIME TO 50% MORTALITY-1,000 Mlnutes FIGURE 25. ';.\1edian times to death at high and low temperatures for young sockeye. Broken lines indicate levels of temperature causing little ot· no mortality for continued exposure. Dotted lines join the appt·oximate points of inflection. pt;, -~-- fl I I I 11 304 Young sockeye are usually lake-dwellers, frequenting the open water and subsurface regions, probably in the cooler temperatures in the vicinity of the thermocline (Ricker, 1937). During the summer they are rarely seen or caught in the shallow, littoral zones oi the lake (Brett and McConnell, 1950) where young coho, shown to have a higher temperature-resistance~ may b~ found on occasion in abundance. The sockeye migrate in the spring of the year shortly after the ice has left more northern lakes, or following rising spring temperatures ~0~ • • 0 .. w a: ::::» 1- <t a: IIJ a. ~ 26 I&J 1- ~ Salvelinus fontinalls~~ -..'::~--~oe-......_-.... e Salmo solar ~,.,-............. ~ ' .......... 1--•il........,_ 0 • ....... " .......................... "' ~~'--.... ,,::·b--·~- 0 i ..... A ........ ..... ..... Cristivomer namayce--'-... ............... o '-......._ -A .... .._oncorhynchus tschowytscha ..... ..... .... ---........... • ---1:!. ... ..... - o ............... 1o Jo. kisutch ......._ '-... O.c;~orbuscha \:'.t..._ 0. nerlla Oncorhynchus keto 10 TIME TO 50'Y• MORTALITY -Minutes FIGURE 26. 1Iedian resistance times for different salmonoids acclimated to 20°C. Data for other than Oncorhynchus from Fry (1947). in such a lake as Cultus which may remain uopen" throughout the year (Foerster, 1937). Termination of the migration is closely correlated with ascending surface temperatures and the presence .of a well established epilimnion which Foerster (1937) points out may form a temperature block. Coho continue to migrate dmwnstream after sockeye have ceased to move. Young pink and churr usually do not experience warm lake or river waters. Their migration to sea almost immediately after hatching in the spring of the year eliminates any such high-temperature experience at this stage of life. Their intolerance to fresh water would probably be more pronounced at higher tem- peratures. The limited success of Chin and Kuroda (1935) in holding churn salmon in fresh water beyond one year depended in part on low temperatures. The interaction of thermal and osmotic stresses requires investigation. The spring salmon, although a first year migrant to sea, may be found during the summer in streams and rivers, and is more like the coho in this res- ( -- siderati bt! the This grou made on Five spring nerka), other, but (4) n·lation tcmperatu (5) the five (6) t!tffering The • _j r I Oi 1l ,• t ~. t i ~· Jgnt I here. ln )r -~ ures 305 pect. These two species were the most tolerant to high temperatures. The problem of their rektive abilities to remain active at high and low temperatures must remain unanswered· for the present. Observations, both field and laboratory, indicate-a greater ability to feed and sport (jumping, darting, etc.) in ·warm waters among the spring and coho than.arnong the remaining three species. On the basis of morphological studies, rates of growth and life history con- siderations, Milne (1948) concluded that the pink and chum salmon appeared to be the most specialized, and the spring and coho probably the most prir.nitive. This grouping of the species within the genus is in accord with distinctions made on the basis of temperature-tolerance. SU::\Il\IARY Five species of Pacific salmon are found in North American w·aters, the spring (Oncorh)!_nchus tschawytscha), the pink (0. gorbuscha), the sockeye (0. nerka), the chum (0. keta) and the coho (0. kisutch). Young of these species, averaging 4 to 5 em. in length and 1 gm. in weight, were used in a series of ex- periments concerning toleranee to high and to low temperatures. Two months after hatching, each stock of .fish was divided into five groups for acclimation to 5°, 10°, 15°,20° and 23°C. (24°C. in spring and pink); acclima- tion and lethal .. temperature experiments continued throughout the following f: ·r months. Resistance times were determined at intervals of 0.5°C. for high _nperatures and L0°C. for low temperatures (0.0°C. and above). Upper lethal temperatures were calculated for exposures of 10,000 minutes (one week) and lower lethals for exposures of 5,500 minutes (approxi"mately four days). RESISTANCE TO HIGH TEMPERATURES. A statistical analysis, using the methods of analysis of variance for test temperatures ranging from 24.5° to 27.5°C. and acclimations of 10° to 20°C., for all species, led to the following conclusions: (1) No significant difference in response to upper lethal levels of temperature exists between spring and coho salmon. (2) Spring and coho salmon show a highly significant difference in response from that of either sockeye1 pink or chum (P < .01). (3) Pink and chum salmon show a barely significant difference from each other, but not from sockeye in either case (P = .05). (4) There is a very highly significant (P < .01) linear fit of the logarithmic relation for these data (logarithm of the median time to death in relation to the temperature causing that death). (5) The slopes of the lines relating log resistance time to temperature for the five species are not significantly different. (6) The same relation can be applied to each species with equal ~..·onfidcnce, differing only in the temperature level at which this relation exists. The ultimate upper lethal temperatures for each species were: spring- 2i5.10C., coho-25.0°C., sockeye-24.4°C., pink-23.9°C., chum-23.8°C. CRITERION OF DEATH AT LOW TE;<.tPERATURES, It was discovered that with the approach of death from a low temperature. the opercula commence to fan out perceptibly:This creterion was shown to be significant when compared with data WW:·~ ~·· , . ··- I l 306 from re(ryvery tests at warmer temperatures. Thus, the individual times to death al: ~:h·en low temperatures could be recorded and median resistance times det.:.ri'i:~~ned wr each sample. RESISTANCE TO LOW TE).lPERATURES. The relation ')etween resistance time to low. temperature and the level of that temperature has not been resolved into a simple equation as has been the case for heat-tolerance relations. A variable but usually sigmoid to double sigmoid pattern characterized the curves ,vhen plotted on normal axes. An initial period of rapidly increasing resistance to low te11,perature was followed by relatively little change in resistance for two and. three degrees increas~ in temperature (15° to 23°C. acclimation). Xo mortality usually occurred within a degree or so above this latter zone for a considerably increased exposure time up to 5,500 minutes. · The young salmon were very sensitive to low temperatures. Among the four species tested, the coho and sockeye salmon could not tolerate long exposure (four days) to 0°C. even when taken from holding temperatures as low as 5°C. The lower lethal temperatures for the highest acclimation, 23°C., were: spring-7.4°C., coho-6A°C.l sockeye-6.7°C.-, chum-7.~°C. 1HXED LETHAL EFFECT OF LOW TE~!PER.-\.TURES. From acclimation tempera- tures of 20°C. and above, mixed responses were noted in the lethal baths. A rapid death of all fish occurred at the lowest temperatures. Temperatures slightly above this level caused rapid death in part of the sampie followed by a long dday and then death of the remainder. Temperatures somewhat higher, yet still low enough to cause death, did so only after prolonged exposure. By plotting the data on probability X logarithmic paper it was possible to discriminate clearly between the two trends of death. Exposure to the same low temperatures in lethal baths containing 9.9%0 sea water instead of fresh \Vater (slightly hyper- tonic) resulted in partially increased tolerance among sockeye salmon. It therefore appears that three causes for death may be involved: one, a very rapid agent usually effecdve 'vi thin GO minutes of exposure, a· second: not so rapid in action, and a third which is related to osmotic balanc·e. SIZE EFFECT. Xo significant difference in the size of the first and last fish to die from high temperatures was present. For death from low temperatures, however, the size distribution of the dead fish from samples in which 30 to 50 per cent of the fish died showed a significantly lower :nean length than in the balance of living fifh. The earlier death among smaller fish appeared to be IJartly the result of grnater susceptibility to "rapid" cold-death. ZONES OF THERMAL TOLERANCE. The zones of thermal tolerance were con- structed graphically and the areas calculated in units of degrees Centigrade squared. The high degree of sensitivity to low tcmperrt.tures, almost conftning these species to acclimation temperatures above 0°C., results in a comparatively low thermal tolerance rating. The spring and coho were almost identical, with 529 and 528 units respectively; the sockeye was ne:<t with 505 unitsj followed by the chum with 4G8 units. Lacking low lethal-tempe~ature data, the pink aalmmt cannot be included, but one preliminary experiment suggested a lower resistance than in the chum s3.lmon, and consequently a smaller zone of tolerance. Even ;:: .aw the arclim CO. t•mpha~. hy pre~ n:fiista than i · is mor salmon: ) GI~ stcnotijl hiokinti the ~lir\ the pint ... ~ taxono1 i BHl.lmR.~, Ul3 Btst~T, r .... ~ , lJt'tl. Bt.ISS, c The' Cal ~ llRETT, j Soc., Ser.,' BCRRO\\ Cull Ctus, %. long D.\\'RS1' org,li O.w, F., l >on)<:R .·. ·. \\ ith men . D\'~toSD, ' 103U I I ---- 307 the most tolerant of the Pacific salmon was considerably below the speckled trout Salvelinus fontinaHs, with a calculated area of G25 ·units. PREFERRED TEMPERA.TURES. Comparatively little difference in preferred temperature was recorded experimentally, either between species or for differences in acclimation amounting to l5°C. in some instances. On the average, no greater difference than 3°C. (le to l4°C.) was displayed behveen means from different acclimations. The region of greatest preference lay in the 12° to l4°C. stratum. Cm.lPARISON OF TE~IPERA.TURE TOLER.\NCE. In making comparisuns, most empl1asis was placed on the resistance times to high temperatures. Experiments by previous investigators .., ... ith other salmono\ds demonstrate a greater heat resistance in two species of Salmo and lesser resistance in Crisli'vvmer namaycush than in the Oncorhynchus group (all acclimated to 20°C.). Sai?Jelinus fontindis is mo1 e resistant to temperatures of 27°C. and above than any of the Pacific sahi'On; however, below t~.is level, the generic distinction does not apply. G ~NERAL CONCLUSIONS. The species of Pacific salmon are comparative!~· stenothermal. An intolerance to low temperatures particularly restncts their biokinetic range. For prolonged exposurf: (up to one week) to high temperatures the spring and coho salmon 'vere most resistant, the sockeye intermediate anti the pink and chum salmon least resistant. These differenc~"s are in keeping with ta.-:onoii1ic conclusions and certain ecological distinctions. REFERE~CES BEr.EHRADEK, J. Temperature and Jiving matter. Protoplasma JionograpMen, 8, 1-229, Berlin, 1935. Br;:>;ET, L., A:::.;-n G. 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'lt.'issett· sclmft. zool., 134, 641-692, 1929. - IIOL , 4. UQ,i ... •.. , ,.. t • 1 \ ·~ \ ' ' t 'i\ i "' lt 1l " " \ l ,. . .• •... :··"· ~--1 LJ r L r1 u ' lr 309 v;Teitere Untersuchungen iiber die Kaltebestandigkeit poikilothermer Wirbeltiere. Zeitscht. f. wisscnsclzajt. Zool., 136, 195-209, 1930. Zur Kaltebestandigkelt poikilothermer Tiere, Untersuchung an Schnecken und Fischen. Biol. Zentralblatt, 56, 301-822, 1936. \VILDER, D. G. A comparative study of anadromous and fresh water populations of the eastern speckled trout (Salvelinus fontinalis). Ph.D. thesis, Univ. of Toronto, 1944. YATES, F. The design and analysis of factorial experiments. Harpenoen: Imperial Bureau of Soil Science, 1937. ---