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Mechanisms involved in the injury and death of fish by chilling temperatures Smith, Frederick Dabell 1950

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(ITI ft I 1 MECHANISMS INVOLVED m THE INJURY AND DEATH OF FISH BY CHILLING TEMPERATURES -by-FREDERICK DABELL SMITH A THESIS SUBMITTED IN PARTIAL FUXE1LIWMT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS HI THE DEPARTMENT OF ZOOLOGY THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER 1950 THE UNIVERSITY OF BRITISH COLUMBIA VANCOUVER. CANADA March 5th, 1951 The L i b r a r i a n , University of B.C. Dear S i r : This is to c e r t i f y that r-Mr. Frederick Dabell Smith has presented an accept-able thesis for the Master's degree and the required standards f o r the thesis have been met. The abstract submitted i s approved. Yours very t r u l y , W.A. Clemens, Head, Department of Zoology W.S. Hoar, Prof, of Zoology & Fisheries 2 . ABSTRACT The behaviour reactions and the mechanisms involved in the injury and death of goldfish (Carassius auratus) exposed to chill temperatures have been investigated. Upon direct transfer to colder water goldfish exhibit an in i t i a l shock reaction followed by a primary chill-coma reaction with the latter reaction sometimes being followed, after apparently normal recovery, by a secondary chill-coma that ends in death. The first two reactions are believed to result from the effect of an excessive thermal stimulation of the cells of the central nervous system whereas the death that follows the secondary c h i l l -coma is attributed to a disruption of the osmotic regulatory system. Within the range of size compared (three to ten centimeters and one to twelve grams), the tolerance to chilling temperatures of gol'dfish of the same or nearly the same age increases as the size of the fish increases. Statistical analysis of this relation shows a significant positive correlation between the survival times and the weight of the fish over their length as well as a significant negative correlation between the survival times and the surface area of fish over their weight. Several factors are thought to be involved in this effect of size of fish on tolerance to chill temperatures, viz., the insulation and surface area of fish relative to body mass in relation to the rate of heat loss and therefore to the body temperature; the g i l l surface area of fish relative to body mass in relation to the rate of abnormal osmotic passage of water subsequent to a disruption of the osmotic regulatory system. 1 ACKNDWLEDSEMENT I wish to express my appreciation to the Head of the Department of Zoology, Dr. W.A. Clemens, for his generous assistance to me during my stay at the university as a graduate student, I also wish sincerely to.thank Dr. William S. Hoar, Professor in the Department of Zoology, \inder whose supervision and guidance the present work was done. The granting of a special research scholarship by The British Columbia Packers Ltd. to sponsor this project is gratefully acknowledged. CONTESTS Acknowledgement • 1 Abstract 2 Introduction 3 Material and Methods • — 22 Experiments and Results General Observations — • 30 The Influence of the Size of Goldfish on their Resistance to Chill-Temperatures 31 Rate of Fall of the Body Temperature of Goldfish on Being Transferred from Warm to Cold Water : 33 Discussion 48 Summary — — — — — — 6 0 References 6 2 INTRODUCTION The response of a homoiothermic animal exposed to oold i s to "bring into effect certain chemical and physical re-actions which maintain the normal constant body temperature ^The physical reaction that occurs in these animals oonsists of a decrease in the blood volume with the blood becoming more concentrated ( l ) , and of a constriction of the cutaneous blood vessels which oauses an eversion of blood from the surface to the internal organs (7)» These two effeots, both of which are brought about by the action of the autonomic nervous system (7), reduce the loss of heat from the body. The chemical reaotion that ooours on exposure to oold oonsists of an increase in the metabolic rate witjj a consequent increase i n the production of heat ( l , 13)«> There has been considerable dispute as to whether this increase in the metabolism i s induced solely through muscular movements, including shivering, or whether other mechanisms suoh as secretions from the adrenal medulla and the thyroid are brought into play as w e l l . Bazett (2) and Morin (39) conclude that reflex shivering or other reflex increases i n movement probably constitute the chief methods by whioh the metabolism is increased in response to cold and that the other mechanisms ealy exert a comparatively small effect. The centre for nervous control of ohemioal regulation against cold, aooording to 4. Ransom (43), i s situated i n the oaudal part of the hypothalamus? This i s influenced reflexly from the skin and d irect ly by the temperature of the blood flowing through the region. In those animals with a well-developed physical regulation, chemioal regulation only appears when the external temperature descends below 14 to 15°C. whereas physical regulation appears as soon as there is; a small decrease in the external temperature; thus, ofaemioal regulation i s regarded as the more primitive type of regulation against the effeot of cold ( l , 7, 13)• On the other hand, when an aquatic poikilothermio animal such as a f i sh i s exposed to oold water temperatures i t s body temperature and therefore i t s metabolio rate declines u n t i l the body temperature approximates the temperature of the surrounding water (4, 5, 11, 19, 28, 30, 44, 46)» The reasons for the inab i l i ty of the f i sh and other afuatio poikilothermio animals to maintain a constant body temperature when the environmental temperature ohangea are threefold. "First, the f ish do not possess the physical regulating meohanisms of the homoiothermio animal and there-for are not able to regulate heat loss . Seoond, the f i sh , unlike the terrestrial poikilotherme (36, 3>, 37), i s surrounded by water whieh has a thermal conductivity that i s about twenty-five times that of dry a i r (2) and i s , therefore, an idea l heat-absorbing medium. This la t ter 5. fact combined with the faot that the blood of the f ish i n passing through the exposed g i l l s i s spread out so as to expose a greatly increased surfaoe to the c h i l l i n g effect of the water, greatly fac i l i ta tes the transfer of heat from the body of the fish to the water (19, 28). Third , the f i sh does not possess the capacity-*- to produce the large quantities of heat capable of being produced by the higher level of metabolism of the homoiothermio animal. The operation of these three factors is such that after a lowering of the water temperature the body temperature of the f i s h decreases and then becomes steady again i n from a few seconds to a few minutes (19, 41). The rate of f a l l of the body temperature i s governed by the difference of the temperature of the water tolthat of the f ish and by the size of the f ish (41). Benediot (j>) and Benedict and lee (6) attribute the lower metabolio and therefore lower heat production level of the poikilothermio animal as being primarily due to a poorer distribution of blood to the outer tissues and peripheries with a resulting ppor supply of oxygen and nutrients to this region. Thus, Benediot and lee (6) show that when the heat production is compared for a g roup of animals such as the rabbit , marmot, tortoise, and snake with a l l being at the same body temperature of 3 7 ° C , that both the heat production as measured in calories and the adequacy of the blood dis-tribution decrease i n the same order as the animals are l i s ted; that is,"where there is the best blood distr ibut ion, there i s the greatest heat production"^). Also , Kidder (28) found that the f i sh whioh possessed a better development of the digestive and circulatory systems had a greater excess of blood temperature o#er the temperature of the environment than did the f i sh with a poorer development of these systems. Kyle (30) , on the other hand, correlates the small production of heat i n the f i sh with the faot that the oxidation of food-stuffs in f i sh i s very o o m p l e t e ( 4 2 ) „ 6 When the temperature of the water Is constant and the heat exohange "between the f i sh and i t s environment has reached i t s equilibrium the "body temperature of the f i sh i s from 0 to 1°C. above the temperature of the water (4, j>. 11, 19, 28, 30, 44, 46). Since f i sh are not in a position to be cooled by the evaporation of water from their body surface and since suoh rapid conduction of heat occurs from the water to the body, tfessr i t is impossible for the body temperature of f ish to be below that of the water exoept i n those oases when there i s a lag of the increase of the body temperature behind an increasing water temperature ( 1 9 ) » Within l imi t s , i t has been noted that the temperature difference between the f ish and the water appears to be inversely proportional to the temperature of the water ( l l ) ; thus, the temperature of the f i sh at the higher water temperatures i s the same or nearly the same as that of the water and at the lower water temperatures the temperature of the f ish may be as much as 1°C. above that of the water. With regard to the measurement of the temperature of f i sh , an interesting observation which seems to have been ignored by later workers was made by Kidder i n 1879 ( 2 8 ) . According to this author, the measurement of the temperature of a f i s h by the insertion of a temperature recording instrument into the intest inal canal i s not a true indication of the body temperature of a f i s h . To quote Kidder: "And, while i t i s d i f f i ou l t to believe that 7 the ohemioal changes neoessary to the nutr i t ion, waste, and repair of the body of a f i sh , taken together with i t s active muscular movements, can go on without the evolution of a large amount of animal heat; i t i s also plain that we are nottfco expect to f ind the manifestation oflthis heat either i n the intes t ina l canal, a mere osmotic tube for the passage and absorption of the food, scarcely vasoular and barely separated from the surrouuding water by the thin bloodless walls of the abdomen; nor in the a r t e r i a l blood returning from the g i l l s , ch i l led down to the temperature of the water with which i t has just been in intimate oontaot*" "We should expect to find the blood of a f i sh at i t s warmest after having been distr ibut-ed to the substance of the body, having furnish.; ed the material for nutr i t ion , taken up the results of waste, and received the heat developed by these processes and by the conversion of muscular motion; that is to say, i n the heart and branohial artery*" Kidder suggested also that the goosefish (Lophius  pisoatorius, Linnaeus) i s especially suited for measure-ments of body temperature sinoe i t i s provided with a very large heart and branohial artery* In any case, under certain circumstances such as unusual physiological aot iv i ty or the absorption of any form of radiation, differences of even one or more degrees centigrade ooour between the internal and external temperatures of f i sh , even when the temperature measurements are made by insert* ion of the temperature reoording instrument into the intest inal oanal (4, 19, 28)• An important active response of f i sh that has been observed onmtheir being transferred to oolder water is that after the i n i t i a l f a l l i n their respiratory metabolism that results from the f a l l in the body temperature there follows 8 an increase i n the metabolism that i s maintained. The opposite e f f e c t has also been demonstrated for f i s h trans-ferred from a lower to a higher temperature; that i s , the increase i n the respiratory metabolism consequent upon the increase i n the body temperature i s followed by a decrease i n the metabolio rate (51, 52). These reactions on the part of f i s h have been demonstrated by Sumner and Wells (52) and by Sumner and Doudoroff (51), both of whom used the rate of death r e s u l t i n g from the use of agents that cause death by asphyxiation to measure the rate of respiratory metabolism* Sumner and Doudoroff (51) also have demonstrated these relationships by a d i r e c t count of the respiratory movements. I t has been found that nearly the maximum change i n the respiratory metabolism occurs a f t e r a period of one day or even less i n the oase of ffundulus parvipinnis and Gil l i o h y t h y s m i r a b i l i s (52),, and f o r the l a t t e r f i s h i t was found also that the respiratory metabolio rate, as measured by i t s resistance to K01T, increases about f i f t y percent i n the f i r s t day with any further increase only being detected s t a t i s t i c a l l y ( 5 l ) » This same physiological adaptation i n the ef f e c t of temperature upon the respiratory metabolism of f i s h to a ohange i n temperature has also been revealed by the faot that when f i s h that have been kept at high and low temperatures are transferred to a common intermediate temperature, that those from the higher temperature display a lower 9. s u s o e p t i b i l i t y to l e t h a l agents that a f f e c t the r e s p i r a t i o n and therefore a lower rate of respiratory metabolism than do the f i s h from the colder temperature (51* 52, 58). Thus, i n one experiment with gobies ( G i l l i o h y t h y s m i r a b i l i s ) , two l o t s of nine f i s h each which had been kept f o r 17 days at approximately 11 °C. and 55«5°C», respectively, were tested a f t e r both l o t s had been held at a oommon, intermediate temperature f o r 48 hours and i t was found that the l o t kept at the lower temperature gave a figure f o r oxygen consumption almost twice as great as the l o t kept at the higher temperature (56). A si m i l a r experiment to th i s one by Sumner and Doudoroff (.51) revealed that this r e l a t i o n s h i p of respiratory metabolic rate continues f o r several days a f t e r transfer to the intermediate temperature. Wells (57) has demonstrated by d i r e c t determination of oxygen consumption i n Fundulus parvipinnis that "the rate of metabolism of fishes at any given temperature i s dependent upon the temperature of the water to which they have been (previously) acclimatized." Behre (5) and Mellanby (25) have noted a s i m i l a r p h y s i o l o g i c a l adaptation i n Planarla dorotoophala and i n Salamandra salamandra L., respectively. In a discussion of experiments i n which the periods of acclimatization ranged from three days to three months, Miss Behre states? "7/orms tested immediately a f t e r they have been l i v i n g f o r a shorter or longer time show greater s u s c e p t i b i l i t y to cyanide than those whioh have been l i v i n g i n d e f i n i t e l y at a higher 10. temperature; those tested immediately a f t e r they have "been put into a lower temperature than that at whioh they have been l i v i n g f o r a shorter or longer time show a lower s u s o e p t i b i l i t y to oyanide than those which have been l i v i n g i n d e f i n i t e l y at the lower temperature." Mellanby found that a salamander acclimatized to 10°C. and placed at 0°C,» had a heart rate approximately twenty beats-to the minute, whereas a salamander acclimatized to 30°C. and placed at the same temperature had a heart rate eight beats or les s to the minute. A relat i o n s h i p of metabolism to body temperature that possibly i s linked with these observations i s that which Pox (17) has reported on the metabolism of a varie t y of marine poikilothermio animals l i v i n g at d i f f e r e n t latitutes; thus, the metabolism of these animals i s such that i t i s higher f o r those l i v i n g i n the cold l a t i t u d e s than f o r those l i v i n g i n the warm latit u d e s when both are compared at the same environmental temperature. Ho suggestions appear to have been made as to the hormonal or nervous mechanism responsible for the increase i n the rate of metabolism following a transfer of these poikilothermio animals to cold temperature water. As well as the physiological response of f i s h noted above on being exposed to cold water temperatures, there i s a biochemical change that i s also* known to occur. This change consists of a decrease i n the degree of saturation and therefore of the melting and s o l i d i f i c a t i o n points of 11 the |ipoids of the f i s h (4, 24). This a l t e r a t i o n of the melting point ooours not only i n the reserve supply of stored f a t but also i n tk# l i p o i d constituents of the protoplasm (52)• The oold temperature apparently influences the formation of a more unsaturated l i p o i d by enhancing the action of the condensation type of fa ^ f o r m a t i o n rather than the reduction type. Conversely higher temperatures favour the action of the reduction raotion with a consequent formation of higher melting point body l i p o i d s (24). Both Belehrgfdek (4) and Heilbrunn (24) promote the b e l i e f that t h i s a l t e r a t i o n of the degree of saturation of the body l i p o i d s plays an important part i n the acclimatization process of f i s h and other animals. In addition to the above desoribed phenomena that ocour when f i s h are exposed to cold temperatures there also has been observed to occur i n the case of some f i s h a shook reaction that ends i n a p a r t i a l or complete ooma~liko atate and whioh i n turn i s eithe r followed by death or by recovery that may be permanent or only temporary (4, 15, 16, 55)• Doudoroff (15) i n experiments with the marine f i s h G i r e l l a nigricans (Ayres) describes the 1 shook reaction as "ranging from mild di s t r e s s and disturbance of equilibriim to a v i o l e n t , oonvulsive paroxysm, • • • ". The coma-Aifee-state that follows the i n i t i a l shock effeot consists of p a r t i a l or complete oessation of respiratory and other movements and Doudoroff (15, 16), following tjie terminology 12 started by Semper (45), refers to i t as "primary chill-coma." Doudoroff noted that when recovery from the primary c h i l l -ooma occurs only temperarily there r e s u l t s a seoond coma-l i k e state i n which there i s again cessation of respiratory movements and a f a i l u r e to respond to stimulation* This l a t t e r state whioh i s followed by death i s fe f e r r e d to as "secondary ehill-ooma." AlthSui^Iitliere have been an abundance of theories suggested to explain the death of f i s h from oold that ocours with ice-formation i n the f i s h , very few authors have suggested theories to explain the death of f i s h by c h i l l i n g , i . e . by cold temperatures above zero degrees centigrade* Be^Lehra*dek (4) and Luyet and Gehenio (33) should be referred to f o r a review of the theories on the cause of the former type of cold death. In the death and i n j u r y of f i s h by the action of oold temperatures above zero degrees centigrade i t i s necessary to, d i s t i n g u i s h between that whioh r e s u l t s from the action of the1 cold being exerted f o r a r e l a t i v e l y long time and that whioh res u l t s from rapid aotion. The mechanism of i n j u r y or death i n the oase of rapid aotion has been suggested by Luyet and Gehenio (33) as being more l i k e l y due to some s t r u c t u r a l changes suoh as p r e c i p i t a t i o n , s o l i d i f i c a t i o n , e t c . Thus, i n explaining the observationsland death by the rapid action of oold above zero degrees centigrade of the Plasmodium of the myxomycete Plysarum plycephalum, they suggest that under the action of 13. r a p i d l y lowering of the temperature, the protoplasmio s o l of the o e l l or c e l l s involved sets to a gel and that t h i s i s a reversible process whioh preoedes "but does not constitute death. In the case of the quick death of f i s h by c h i l l i n g , Weigman (54) contends that t h e i r death on exposure to low temperatu©s i s caused by respi r a t o r y disturbances due to a direot e f f e c t of cooling upon the respiratory centre of the brain. The observation that the f i s h oould be revived a f t e r return to normal temperatures by the app l i c a t i o n of a r t i f i c i a l r e s p i r a t i o n when warming alone was not s u f f i c i e n t i s c i t e d by Weigman as being proff i n favour of his contention. The mechanism of i n j u r y or death i n the case of a slow action of oold above zero degrees centigrade has been suggested by Luyet and Gehenio (33) and by Heilbrunn (24) as being more l i k e l y due to some disturbance i n the i n t e r -play of the phys i o l o g i c a l funotions of the body. Most of the theories that have been proposed to explain the slow type of oold injury or death by c h i l l i n g have been con-cerned with some such phy s i o l o g i c a l disturhanoe i n t h e i r explanation. Thus, the theories of the older and more modern workers, as reviewed by Belehraclek (4) ijji' 1935, are of t h i s type. Aooording to thi s review of B<flehrtfdek»s the more important suggested causes of slow death by o h i l l i n g oan be l i s t e d as follows. 1. disproportion between the v e l o c i t i e s of several v i t a l organic functions; most of the theories of t h i s type have been suggested i n reference to death by o h i l l i n g of plants; 2. changes i n the v e l o c i t i e s of the in t e r r e l a t e d ohemioal reactions of the c e l l ; thus, a number of authors oited by Belehradek (4) propose that "ohanges i n the c e l l u l a r metabolism, i n the ohemioal composition of the protoplasm and of i t s produots, i n the s o l u b i l i t y and i n the absorption of various substances, i n the ohemioal equilibrium inside the c e l l s , i n the r e l a t i v e v e l o c i t y of catenary biochemical processes, e t c are responsible f o r the cessation of v i t a l a c t i v i t i e s by oold and f o r death by ohilling»(4); 3» accumulation of toxic produots whioh at normal temperatures are burnt or eliminated; 4. s o l i d i f i c a t i o n of protoplasmic f a t s ; t h i s suggested oause follows from the faot that the melting point of the protoplasmic l i p o i d s varies with the temperature of acclimatization and as stated by Hammond (22) that f o r fat to be of use as a source of energy i n the body i t must be just f l u i d at the natural body temperature; J5. increase i n protoplasmic v i s c o s i t y or of that of oertain protoplasmic phases; t h i s suggestion i s made by Belehraaek (4) himself since he believes that 1 5 . "suoh an increase would considerably binder free movements of reacting molecules with the r e s u l t that the bioohemioal reactions i n the c e l l would be brought to a s t a n d s t i l l . " Belehradek (4) l i n k s 4 and 5 together by pointing out that the V i s c o s i t y of protoplasm must be expeoted to vary with the melting point of the protoplasmic f a t t y constituents. In addition, i t i s not inconceivable that the ohanges l i s t e d as 2 and 5 would evolve as a consequenoe of the aotion of 5» A more reoent suggestion as to the cause of death by o h i l l i n g of f i s h has been made by Doudoroff ( l6) i n working with marine f i s h , Fundulus parvipinnia. He found that increasing d i s t r e s s indicative of cumulative i n j u r y was aooompanied by loss of tissue water through osmosis f o r Fundulus which were dying i n sea water at slowly l e t h a l low temperatures. In one experiment an average loss of more than twenty-three peroent of the normal water content occurred. When the Fundulus was tested under the same conditions except that f o r t y - f i v e percent sea water was used, the f i s h did not appear to be dehydrated and they survived almost twice as long when the temperature of exposure was 4°0. as did the f i s h i n normal sea water. In explanation of h i s r e s u l t s Doudoroff ( l6) suggested that the primary cause of i n j u r y and death was the injurious effect of cold on the tissues involved i n osmotio regulation (e.g., of the g i l l s , the oentral nervous system and of the integument) with a 16 consequent "breakdown i n the osmotic regulative functions suoh as the active excretion of s a l t s by the g i l l s or the ingest-ion of sea water which ut^er normal conditions compensate for the passive exosmotio loss of water through the external membranes ( 2 7 , 2 9 , 4 9 , 50). Thus, the result of oold i n j u r y to the normal osmotic regulative mechanisms f o r the f i s h present i n the normal sea water w%s gradual dehydration of the f i s h followed by death; however, i n the oase of the f i s h present i n the approximately isomotio medium of f o r t y - f i v e percent sea water dehydration did not occur and therefore death must have been caused by some other less rapid l e t h a l disturbance. D i l u t i o n of the medium made no s i g n i f i c a n t difference i n the oase of death of Fundulus by rapid o h i l l i n g . Doudoroff concludes that osmotic regulation f a i l u r e i s only one of a number of possible causative factors which should be taken into consideration i n further studies of the phenomena of c h i l l i n g * Aside from the explanation of the meohanisms that cause death of f i s h by slow c h i l l i n g there are c e r t a i n factors that are known or suspeoted to a f f e c t the s u r v i v a l time and resistance or toleranoe of f i s h on being exposed to o h i l l i n g temperatures. The more important of these factors are the acclimatization temperature, the physiological condition, and the age of the f i s h . A series of prominent workers, including loeb and Wasteneys (310, Huntsman and Sparks ( 2 6 ) , Hathaway ( 2 3 ) , 17 Wells(580, Doudoroff (14), Sumner and Doudoroff (51), Brett (8, 9, 10)., Fry, Brett and Clawson ( l8 ) , and Doudoroff ( l 6 ) , nave fi r m l y established the faot that acclimatization ao does exert an* influence upon temperature tolerance to high and low temperatures of both fresh-and salt-water f i s h . Thus, within l i m i t s , acclimatization of a f i s h to a low temperature increases i t s toleranoe to s t i l l lower temperatures, whereas acclimatization to a high temperature increases i t s toleranoe to s t i l l higher temperatures. Conversely, acclimatization of a f i s h to a low temperature decreases t h i s tolerance to high temperatures whereas aoclimatizatio# to a high temperature deoreases th i s toleranoe to low temperatures. Acclimatization to c h i l l i n g temperatures i s r e l a t i v e l y slow and the resistance i s l o s t no more slowly that i t i s acquired. Thus, Doudoroff (15) states that " a f t e r any given r i s e or f a l l of environmental temperature within the normal range, about f i f t y percent of the t o t a l r e s u l t i n g change of oold tolerance occurs i n two days, and oomplete acclimatization ( i . e . , constant cold-tolerance) i s apparently achieved i# about twenty days." On the other hand, aoolimatization to high temperatures i s gained r e l a t i v e l y r a p i d l y a f t e r a r i s e of temperature and l o s t slowly a f t e r cooling ( 1 5 ) « Thus, Hathaway (23) fou^d that the greater part of the aoolimatization effeot of heat to be oomplete within the f i r s t day; also, Sumner and Doudoroff (51) found that when 18. f i s h were transferred from 20°C. water to 30°C. water that even a h a l f - hour stay i n the 30°C. water l e d to a consider-able increase i n tolerance to high temperatures, and this inorease continued gradually up to ten days. Fry, Brett and Clawson (18) established the r e l a t i o n between a number of f i f t y - p e r c e n t - l e t h a l c h i l l temperatures and the temper-atures of acclimatization as well as the r e l a t i o n between a nunjber of f i f t y - p e r c e n t - l e t h a l high temperatures and the temperatures of acclimatization (see f i g u r e s 1 and 2 of Fry, Brefct, and Clawson (l8)) f or gold f i s h (Carrasius  suratus L.) that averaged two grams i n weight. They also found that a difference of three degrees i n the acclimatizat-ion temperature resulted i n about a two degree change i n the f i f t y - p e r c e n t - l e t h e l c h i l l temperature and i n a one degree change i n the fifty-peroent-lethai high temperature. Although not much studied a few suggestions have been offered as to the mechanism involved i n acclimatization of f i s h to temperature. Davenpofct (12) suggested that the inorease i n tolerance to heat might be caused by a lowering of the water oontent of the protoplasm with a consequent r a i s i n g of the temperature necessary to cause coagulation. Loeb and Wasteneys (31) compared acclimatization with the annealing of glass. As noted e a r l i e r , Belehradek (4) and Heilbrunn (24) both support the contention that acclimatizat-ion to temperature, at least to high temperatures,. involves a change i n the degree of saturation and therefore of the 19. m e l t i n g and s o l i d i f i o a t i o n p o i n t s o f t h e l i p o i d s o f t h e f i s h . I n t h e oase o f a o o l i m a t i z a t i o n t o c h i l l i n g t e m p e r a t u r e s D o u d o r o f f ( l 6 ) , i n d i s c u s s i n g t h e mechanisms o f a c c l i m a t i z a t i o n , i n d i c a t e d t h a t t h e c h a n g e s i n v o l v e d i n t h e a c c l i m a t i z a t i o n t o c o l d a n d i n t h e a c c l i m a t i z a t i o n t o h e a t a r e more o r l e s s i n d e p e n d e n t o f one a n o t h e r . He c i t e s i n s u p p o r t o f t h i s i n d i c a t i o n t h e f a c t t h a t he o b s e r v e d i n G i r e l l a j k g r i o a n s ( A y r e s ) t h a t a change i n c h i l l t o l e r a n c e c o u l d be o b t a i n e d w i t h o u t a c o r r e s p o n d i n g change i n h e a t -t o l e r a n c e and t h a t t h e r a t e s o f change o f r e s i s t a n c e t o h e a t a n d t o c h i l l t e m p e r a t u r e s a r e q u i t e d i f f e r e n t . V e r y l i t t l e i s known, t h e r e f o r e , o f t h e mechanisms i n v o l v e d i n t h e a c c l i m a t i z a t i o n o f f i s h t o c h i l l t e m p e r a t u r e s , a l t h o u g h some t h e o r i e s have b e e n put f o r t h i n e x p l a n a t i o n o f a c c l i m a t i z a t i o n o f f i s h t o f r e e z i n g t e m p e r a t u r e s . F o r a r e v i e w o f t h e l a t t e r see B e l e h r a d e k ( 4 ) , L u y e t and G e h e n i o ( 3 2 ) , a n d H e i l b r u n n ( 2 4 ) . W e l l s ( 3 5 ) f o u n d t h a t t h e p h y s i o l o g i c a l c o n d i t i o n o f t h e f i s h j u s t b e f o r e t h e b r e e d i n g s e a s o n c a u s e d g r e a t e r r e s i s t a n c e o f t h e f i s h t o o c h a n g e s o f t e m p e r a t u r e t h a n t h a t w h i c h o o o u r r e d i m m e d i a t e l y f o l l o w i n g t h e b r e e d i n g season« A l s o , i t i s q u i t e p r o b a b l f t h a t a p o o r l y n o u r i s h e d f i s h w i l l be l e s s t o l e r a n t t o low t e m p e r a t u r e s s i n c e i t h a s b een shown t h a t d u r i n g f a s t i n g o f t h e A f r i c a n l u n g - f i s h ( P r o t o p t e r u s a e t h i o p i o u s H e c k e l ) tfes*t t h e oxygen consumption and m e t a b o l i c r a t e d r o p o f f ( 4 7 , 4 8 ) . I n t h e 20 oase of we11-nourished Fundulus parvipinnis, however. Wells (57) has found that during the sdoond to seventh day of starvation the oxygen consumption of the f i s h e s remains very constant; therefore, these f i s h might not at f i r s t show a decrease of tolerance to low temperatures* The influence of the age of a f i s h on i t s tolerance to c h i l l i n g temperatures i n the f i e l d ( i . e . , i n the ocean and i n lakes) and i n the lab has been reported on by several authors (20, 21, 25, 55). Thus, i n the l a b . Wells (55) observed that small (young) oyprinid f i s h were l e s s affected by the change from warm to cold temperatures than we're adults of the same species. Gunter (21) found that under natural conditions the larger (older) specimens of certain species of fishes were k i l l e d by oold waves i n proportionately greater numbers than the smaller (younger) specimens. Other workers (20, 25) also have observed that under natural conditions the young of several f i s h e s seemed better able to withstand than t h e i r elders the e f f e c t of c h i l l i n g temperatures* Belehradek has stated that the age of a f i s h i s the most important i n t e r n a l factor involved i n c h i l l i n g (4). Ho references have been found i n the l i t e r a t u r e on the influence of size i n i t s e l f on the tolerance of f i s h to c h i l l i n g temperatures* The following experimental work, therefore, as well as being concerned with the behaviour of g o l d f i s h exposed to o h i l l i n g temperatures also includes a 21. determination of the influence of the size of f i s h on t h e i r resistance to c h i l l i n g temperatures with the hope that such information might a i d i n revealing the mechanisms involved i n the i n j u r y and death of f i s h by c h i l l i n g temperatures. 2 2 . j MATERIALS AND METHODS The fish used for these experiments were goldfish (Carassius auratus) varying from three to ten centimeters in length and from one to twelve grams in weight and were purchased from Goldfish Supply Company, Stouffville, Ontario. The conditions under which the gold-fish were kept at Stouffville are not known to the author. In the laboratory the goldfish were kept in glass aquaria containing fresh tap water. The water of the aquaria was maintained at the desired constant temperature by the use of a Fenwal thermoregu-lator and Lo-lag Immersion heater. The water was aerated by a stream of compressed air. A chill temperature was selected that would be lethal for fifty or more per cent of the fish but which would act slowly enough to give a measurable spread in the survival times. This selection was necessary so that the influence of the size of fish on their resistance to chill temperatures might be determined and, at the same time, permit the reactions that occur when fish are exposed to chilling temperatures to be observed. The fish were acclimatized to a constant temperature in accordance with the practice of Fry et al (18); that is, the fish were acclimatized at "the rate of roughly one degree centigrade per day." The relationship of Fry et al (18) was then used to select the appropriate ch i l l temperature. In preliminary experiments it was difficult to determine when a fish was really dead and not just apparently so. Thus, there was a persistence, long after a l l spontaneous movements had ceased, of 23. ready responsiveness to sharp mechanical shocks such as pressure at the base of the caudal fin, and momentary (two to three seconds) removal of the fish from the water. Sumner and Doudoroff (51) observed the same behaviour in their chilling temperature experiments with goby fishes (Gillichythys mirabilis Cooper). Further, i t was found that, even after the goldfish became unresponsive to sharp mechanical shocks, they revived when placed in aerated, slightly warmer (5 to 8 C. warmer) water. To overcome these difficulties i t was deemed advisable to em-ploy the non-response to some numerically measurable constant stimulus that coincided with the inability of the fish to be even temporarily revived. For this purpose stimulation by means of a non-polarizing electric current was adopted with at least a thirty second interval being allowed between successive stimuli. The apparatus used, an adaptation of that used by Elson (16a), is shown in Figure 1. The source of electric current is an "Electrodyne Stimulator". On dis-charge of the stimulator the electric current is carried from the non-polarizable zinc-zinc chloride electrodes in the glass beakers through the wooden brides to the water contained in the twelve inch shallow glass bowl. Before use, the wooden bridges are soaked in a one percent sodium chloride solution until they become saturated. The passage of the current through the water results in an electrical stimulation of the goldfish present there. The strength of the stimulus is varied by alteration of the dial on the stimulator. The water of the glass bowl was kept at the same chill temperature that was selected for the fish. 24 Figure 1, Apparatus f o r the Stimulation of Goldfish with a Non-polarizing E l e c t r i c Current,, A: zinc electrodes immersed in 200 c c e of 0.1 N ZnCl2; B: wooden bridges that have been soaked in one percent sodium chloride u n t i l they become saturated; C: beakers containing the ZnCl2 solution; D: copper wire; E: "Electro-dyne Stimulator"; F: f i s h ; G: twelve-inch, shallow glass bowl containing cold water. 25. After the fish became non-responsive to sharp mechanical shocks further exposure of the fish resulted in the strength of electrical stimulus that was required to elicit a response increasing until finally the fish would no longer respond (see Figure 2). When goldfish that had not been exposed to chill temperatures were tested in the apparatus shown in Figure 1 it was observed that the strength of stimulus that was required to elicit a response varied considerably and appeared to be dependent upon the activity of the goldfish at the time of the stimulus; thus, the strength of stimulus required was much increased i f the fish were active at the time of the stimulus. In some instances, goldfish exposed to chill temperatures exhibited this same type of relationship (see Figure 3). As a matter of record, a representative sample of the data on which these observations were made is presented in Table 1. The non-response of a twitch of the fins or of the t a i l of the chill-exposed goldfish to the maximum available strength of stimulus (250 volts) was selected as the end-point for death of the fish, even although further increase in the strength of the electrical stimulus might have elicited a response. Admittedly, this method of selection of the end-point for death was arbitrary, being governed by the maximum capacity of the apparatus. At least, of three goldfish tested, the non-response to the selected end-point strength of stimulus was accompanied by an inability of the three fish to be even temporarily revived in warmer water with artif i c i a l respiration. It is true that the death of the fish, i f death be defined as the attainment of a 2? . 2 6 . Table I; The Strength of Electrical Stimulus (Voltage) Required to Elicit  a Response at Different Time Points for Goldfish Exposed to Chilling Temperatures(exp. A) Fish Ho, Time (hours) and Voltage Readings 3 Time : Voltage: 5.8 30 9.7 100 11.0 U 0 11.4 250 7 Tiflte : Voltage: 8 , 1 13.0 50 70 14.2 250 8 Time : Voltage: 6 .4 11.1 40 40 12.0 320 12.5 150 13.5 250 Time : 8.7 11.5 12.Z 13.5 14.5 14.9 9 Voltage: 30 160 120 80 100 250 Time : 6\8 10.8 12.8 13.7 14.5 15 .1 16.3 13 Voltage: 20 130 150 180 220 150 250 Time : 8 . 1 10.4 11.7 30 Voltage: 30 100 250 Time : 7.2 10.4 10.8 11.5 33 Voltage: 40 80 80 250 Time : 6 .1 7.9 10.1 12.3 12.9 14.0 14.5 14."o" 15.1 1! 34 Voltage: 50 20 100 120 130 120 180 160 110 ; 36 Time : 8.7 9.0 10.4 10.Z 11.0 11.9 Voltage: 40 50 100 50 60 250 37 Time : Voltage: 7.1 30 10.5 170 10.8 170 11.7 250 28 Time : Voltage: 5.3 30 6.2 20 7.0 40 7.5 40 8.1 40 8.4 40 9.2 80 9.7 250 Time : 12.5 12.9 13.2 U . l 15.0 20 Voltage: 60 70 100 110 250 F i g u r e 2 . An example of the r e g u l a r i n c r e a s e i n the s t r e n g t h (voltage) of st i m u l u s r e q u i r e d to e l i c i t a response of g o l d -f i s h exposed to c h i l l i n g temperatures. F i g u r e 3 . An example of the i r r e g u l a r i n c r e a s e i n the s t r e n g t h ( v o l t a g e ) of stimulus r e -q u i r e d to e l i c i t a response of some of the g o l d f i s h exposed to c h i l l i n g tempera-t u r e s . 27. f i i i i I i i i i l i i " * * f O 5 10 15 20 25 Time of Exposure in Hours Figure, 2 • . . . . i . . . . i . . . . I . . . . I . . r t j O 5 10 15 20 20 Time of Exposure in Hours Figure 3. 28. condition in the animal that coincides with the inability of the animal to be revived, may have coincided with the non-response to a smaller or greater strength of electrical stimulus than that used here. Unfortunately, this fact was not determined, but since it was observed that the terminal increase in the strength of electrical stimuli up to the strength of stimulus selected as the end-point occurred within a relatively short time, then the time error involved in relation to the length of the survival time was not large. With regard to this difficulty in the selection of a suitable end-point, Doudoroff (15) states the following: "The accurate quantitative comparison of relative tolerance requires the adoption of a uniform and con-venient 'criterion of survival*, that is an end-point after which the organism is regarded as having succumbed to the effects of the lethal agent. It is best to adopt as an end-point the permanent (not spontaneously reversible) cessation of some important function which is among the first to be arrested. The time required for the attainment of an advanced stage of necrobiosis is not as accurately indicative of relative rates of injury, and may be greatly influenced by temperature and other factors independently of the rate of injury. The permanent cessation of respiratory and other move-ments, either spontaneous or induced by mechanical stimulation, presumably resulting from the functional inactivation of the central nervous system, apparently satisfies the above requirements.H Doudoroffs reasoning might be quite correct but i t is felt that i t is unlikely that any possible deficiencies suggested by him are serious enough to invalidate the results obtained in these experiments. 2 9 . In the preliminary experiments the fish were transferred directly from their acclimitization temperature into a large constant-temperature, aerated, fresh-water tank. The temperature of the water in this tank was the temperature that was lethal for fift y percent of the goldfish of Fry et al (18) acclimatized at the same temperature. In later experiments the acclimatized fish were placed in the same large water tank, the water temperature of which was at or within one or two degrees of the acclimatization temperature of the fish. The water in this tank was then gradually cooled by the refrigeration unit of the tank down to the selected chill temperature. The rate of cooling was between two to three degrees centigrade per hour. The length of time that the fish were able to remain alive under the conditions of the experiment was noted. Also, the behaviour reactions and activity at different temperatures were observed. The weight and the length of -each fish were recorded at the beginning of each experiment. The rate of the decline of the body temperature of goldfish when transferred directly to a lower environmental temperature was measured. A small Taylor mercury-type thermometer having a bulb diameter of 3 millimeters and graduated in degrees Fahrenheit was inserted as far down the gullet of the fish as possible without in-juring the fish. With the thermometer s t i l l inserted in its gullet, the goldfish was transferred directly to the chill-temperature water where i t was held totally submerged with tweezers while the rate of decline of the temperature of the fish was recorded. 30. j EXPERIMENTS AND RESULTS General Observations. In a series of four preliminary experiments, involving a total of one hundred and thirty goldfish, the fish were transferred directly from the acclimatization temperature to the selected chill temperature. In this series i t was observed that the behaviour response was not unlike that described for marine fish by Doudoroff (15, 16) as ''initial shock", "primary chill-coma", and "secondary chill-coma". Thus, upon being transferred to the chill temperature, the goldfish jumped out of the water, showed loss of equilibrium by swimming upside down or on their sides and raced about until they finally came to rest on one side at the bottom of the tank. It was noted that the fish did not immediately or al l together exhibit this unusual behaviour, but that the reaction set in only after a lapse of time and that the length of this lapse of time was longer with larger size of goldfish. In some cases the very small fish almost immediately settled on one side at the bottom of the cold-water tank, whereas for the rest of the fish the lapse of time that occurred before the ini t i a l shock reaction set in varied from a few seconds to almost four minutes. This violent, convulsive paroxysm was then followed by a coma in which there was, depending upon the severity of the chill temperature, a partial or complete cessation of respiratory and a l l other movements. In some of the experiments the chill temperature was very low in relation to the acclimatization temperature and the fish did not even recover temporarily. When the chill temperature that the fish were transferred 31. to was less extreme, they exhibited partial or complete recovery from the coma. Usually i f they showed any signs of even partial recovery the goldfish did so before an hour. When recovery did occur i t came about slowly with respiration being resumed f i r s t , followed by spon-taneous movements and the assumption of an upright position together with a normal appearance and behaviour. When the fish succumbed again after recovery from the primary chill-coma the observed train of events that led up to the death of the fish were inactivity, loss of equilibrium, lack of response to sharp mechanical shocks, and lack of response to a regularly increasing strength of electrical stimulus. The lapse of time that occurred between the end of the primary c h i l l -coma state and the beginning of the secondary chill-coma was quite variable and often quite prolonged, i.e., from three to twenty hours. It was not possible in this series of preliminary experiments to compare accurately the effect of size of the fish on its survival time since special means had not been devised at thi3 stage for ascertaining the end-point of death. However, although by no means decisive, the observation was made that there was a definite tendency in these experiments for an increase in the size of the goldfish to correspond to an increased resistance to chill temperatures. In the two experiments, A and B, in which the fish were gradually cooled from their acclimatization temperature, the goldfish succumbed permanently and exhibited only the secondary chill-coma and not the in i t i a l shock and primary chill-coma reactions. The Influence of the Size of Goldfish on their Resistance to Chill  Temperatures, 3 2 . Experiment A. In this experiment thirty goldfish of varying sizes but a l l within forty-two days of one age (approximately one year old) were em-ployed. The fish had been acclimatized from 17°G. to 24°G. over an eight day period. The fish were placed in 24°G. water and the water was then cooled at roughly 2®G. per hour down to between 1 and 2°G. where the temperature of the water was maintained for the remainder of the experiment. Using the procedure outlined in Methods and Materials the survival times of the fish were determined. The results are presented in Table II and graphs 1 to IV inclusive. The values for surface area used in the relationship of weight over surface area were calculated by use of the formula 10 where W is the weight of the goldfish. The statistical significances of the results are given on in Table III while the "line of best f i t " is plotted the graphs. Experiment B. In this experiment eighteen goldfish of varying sizes but, as in experiment A, a l l within forty-two days of one age (approximately one year old) were employed. The same procedure as in experiment A was followed. The results are presented in Table IV and graphs V to VIII inclusive. The statistical significances of the results are given in Table II while the "line of best f i t " is plotted on the graphs. The text Goulden (18a) was the source of the methods of statistical analysis employed. Examination of the tables and graphs for these two experiments reveals the existence of a positive correlation between the size of 33. goldfish and their survival times; that is, the tolerance of goldfish to chill temperatures increases as the size of the fish increases. In addition, i t is demonstrated that a positive correlation exists between the survival time of goldfish and weight over length and weight over surface area. These latter two relationships are calcu-lated in an attempt to discover some of the factors involved in this influence of size on the tolerance of fish to chill temperatures. Rate of Fall of the Body Temperature of Goldfish on Being Transferred  from Warm to Cold Water. Experiment C. In this experiment four goldfish acclimatized to approximately 23°C. or 74°F. and of varying size were employed. The fish were transferred from water at the acclimatization temperature of the fish directly to water of approximately 2°C. or 35.5°F. The method of taking the temperature of the fish has been outlined in the Materials and Methods. The rate of decline of the temperature of the fish was recorded. The rate of f a l l of the temperature of the thermometer itself after transfer from the warmer to the colder water was also determined. The results are presented in Table V and graph IX. Examination of the results demonstrates that the rate of f a l l of the body temperature of goldfish decreases slightly as the size of fish increases. 34. Table II: Experiment At The Survival Times of Goldfish Exposed to Chill  Temperatures. NO. LENGTH...... WEIGHT WEIGHT WEIGHT . . SURVIVAL TIME LENGTH SURFACE AREA cm. g. hours 11 ... 3.3 1.1 0.33 0.1027 9.3 2. 3.7 1.5 0.41 0.1111 5.9 3 4.4 2.0 0.45 0.1197 11.4 6 3.2 1.1 0.34 0.1027 7.7 7 4.5 5.3 1.08 0.1541 14.2 8 5.0 4.1 0.82 0.1443 13.5 9 4.5 7.6 1.69 0.1692 14.9 11 4.8 5.0 1.04 0.1519 13.6 12 6.6 8.6 1.30 0.1748 18.1 13 6.9 •' 10.9 1.58 0.1863 16.3 15 6.7 9.6 1.43 0.1801 21.0 16 6.5 6.8 1.05 0.1646 17.2 17 5.8 ' 7.7 1.33 0.1699 18.8 18 7.5 11.3 1.50 0.1881 17.0 19 6.8 11.0 1.62 0.1867 20.1 20 5.9 6.8 1.15 0.1646 15.0 21 6.0 6.4 1.06 0.1624 20.2 22 7.0 11.6 1.66 0.1895 17.0 23 6.5 9.5 1.46 0.1792 16.0 24 4.7 3.2 0.68 0.1350 13.1 35. NO. LENGTH cm. WEIGHT g. WEIGHT LENGTH WEIGHT SURFACE SURVIVAL AREA hours 25 . 5.5 , 5.7 1.04 0.1574 15.6 28 3.8 1.9 0.50 0.1180 9.7 30 3.6 1.1 0.31 0.1028 11.7 31 5.9 6.7 1.13 0.1642 18.6 32 5.4 4.8 0.88 0.1505 10.7. 33 4.7 3.3 0.70 0.1363 11.5 34 3.9 1.9 0.49 0.1180 15.9 35 3.6 1.7 0.47 0.1148 11.0 36 4.0 1.9 0.47 0.1180 11.9 37 3.7 1.7 0.46 0.1148 11.7 36. GRnPHI: LENGTH OF FISH PLOTTED FIG BIN ST SURVIVAL TIME l o IS 4 0 3 U R V I V H L T I M E I N HOURS Y 37. GRAPHS'- WEIGHT OF FIffH PLOTTEJD FlGRIN ST SURVIVAL TIME X. f * * m ' ' * O S I O 15 A O 215 S U R V I V R L T I M E , I N H O U R S GRRPHIH: WEIGHT O V E R LENGTH OF FISH RGAINST SURVIVAL TIME I I GRRPHST: L/EI CWT OVER SURFACE BRER OF FISH AGAINST SURVIVAL Table III; Statistical Significance of the Positive Correlation between Length, Weight, Weight over Length, and Weight over Surface Area of Goldfish and their Survival Times when Exposed to Chill Temperatures EXPERIMENT LENGTH WEIGHT WEIGHT WEIGHT LENGTH SURFACE AREA cm, g. w-fcH "V "t" "t" "t" "t" P,05 P,05 P,05 P.05 2.1 1.0 7.0 3.4 13.2 6.4 7.6 3.7 3.7 1.7 4.7 2.2 4.5 2.1 5.0 2.4 o Table IV t Experiment B: The Survival Times of Goldfish Exposed to Chill  Temperatures. NO. LENGTH WEIGHT WEIGHT WEIGHT SURVIVAL TIME LENGTH SURFACE AREA cm. g. hours 2. 5.5 4.8 0.87 0.1505 5.5 3 5.5 5.0 0.91 0.1519 4.3 4 7.2 8.8 1.22 0.1760 5.9 5 7.5 11.3 1.51 0.1880 5.9 6 5.3 7.6 1.43 0.1692 5.3 8 6.0 7.0 1.16 0.1658 6.1 10 6.0 11.5 1.91 0.1885 7.9 11 4.6 3.0 0.65 0.1333 2.9 14 5.0 3.9 0.78 0.1423 4.3 15 5.5 5.8 1.05 0.1580 5.5 16 4.9 3.7 0.75 0.1026 3.5 17 6.6 9.3 1.41 0.1788 8.0 18 5.1 3.1 0.61 0.1342 3.1 19 6.3 8.1 1.28 0.1723 4.6 20 7.1 10.9 1.53 0.1863 12.1 21 5.2 4.8 0.92 0.1504 4.5 22 4.4 2.4 0.54 0.1256 4.3 23 4.9 4.0 0.82 0.1433 4.2 U2. GRRPH "ST* LENGTH OF FISH PLOTTED fl CHIN ST SURVIVRL T l f lE X 8r LU h-U l Z Li U Z * & 3 'LXWC OF BEST FIT": IL 6 h fo Ya. S U R V I V R L T I M E y I N H O U R S ^ 4 3 . GRAPHS! WEIGHT OF FISH PLOTTED RCHINST SURVIVAL TIME g iol e o M »-5« M M L I M £ O F B E S T F X T " s S U R V I V A L T X H C Z N H O U R S I O Y SORVJVBLTine III HOURS y CRRPHDH'.WEICHT OVER SURFRCE RRER OF FISH RGAINST SURVIVAL Table V: Experiment Ct Changes in the Body Temperature of Goldfish of Varying Size when Taken from the  Temperature of 74^F. and Transferred to Another Temperature of 35.5°F. NO LENGTH cm. WEIGHT g. Time (in seconds) and Temperature (in degrees : Readings Fahrenheit) 1 7.1 10.9 Time i Temp: 9 70 10 66 16 64 22 62 27 60 31 58 39 56 44 54 51 50 59 48 73 46 87 44 103 129 42 40 161 38 205 37 231 36 2 4.7 3.2 Time: Temp: 0 70 2 68 4 64 6 60 7 58 9 54 11 52 31 42 42 40 60 38 80 37 110 36 3 4.8 3.1 Time: Temp: 0 70 2 68 4 64 7 64 9 62 13 60 16 56 19 54 21 52 24 50 27 48 29 46 38 44154 44 42 40 73 38 123 36 4 5.6 6.0 Timet Temp: 0 70 4 68 7 66 10 64 14 62 17 60 20 58 27 56 32 54 37 52 41 50 48 48 56 64 75 46 44 42 92 40 121 38 147 37 170 36 Thermometer Time: 0 Ten»p» 70 3 60 5 50 7 44 10 40 12 38 18 29 36 35.5 42 35.5 o-VP v» lf» u» ^ 4 8 . DISCUSSION The lapse of time that occurs before a reaction sets in and the nature of the response of the fish on being directly transferred from warm to quite cold water suggests an involvement of the central nervous system. Doudoroff (16) came to the same conclusion. This observation is in agreement with the statement by Heilbrunn (24) that cold can act as a stimulus and as an anaesthetic. By applying the calcium-release or colloidal chemical theory (24) as used by Heilbrunn (24) to explain the response to stimuli of a cell and by assuming, in line with Weigman's (54) observations, that the cells most vitally affected by the sudden change of temperature be those of the respiratory centre of the brain, a reasonable hypothesis can be formulated to explain both i n i t i a l shock and primary chill-coma resulting either in death or followed by recovery. Thus, by application of this theory to the sudden action of cold, the first action of the cold would be to cause the release of calcium from its protein-binding complex in the cortex of the affected cells with a consequent liquefaction or decrease in the viscosity of the cortex. This freed-calcium would then pass to the interior of the cell where it would cause a brief transitory liquefaction followed by a reversible gelation or clotting of the interior protoplasm that results from calcium-produced surface precipitation reactions. With this clotting reaction there would occur an increased irritability or stimulation of the affected cells. If the cold is severe enough i t would cause the release of calcium in the cell interior as well and thus prevent the internal clotting reaction. 49. This prevention of gelation or clotting both in the cortex and interior of the cell would result in anaesthetization of the affected cells. When the temperature change is very severe then the cold would probably cause the release of so much calcium that the cold would be powerless to prevent clotting in the interior: thus, the excessive stimulation of the cold could lead to complete irreversible clotting, causing in-jury and death to the cells. This calcium-release explanation *H4 which is not too unlike the explanation given by Luyet and Gehenio ( 3 3 ) (see Introduction) can also be related to Weiginan's ( 5 4 ) observation that sudden cold results in respiratory failure of the exposed fish by postulating that the cells most vitally and severely affected would be those of the respiratory centre of the fish brain. Thus, the cold stimulation of this and other parts of the central nervous system would result in the violent activity of the ini t i a l shock reaction; the cold anaesthization of the cells of this same system would produce the primary coma; and i f the cold were severe, its action in causing irreversible injury or death of these cells would result in the death of the fish. In those cases where the cold is not intense enough to cause injury or death of the affected cells then the primary coma would be followed by the recovery of the fish. Since even in the largest fish employed in these experiments the temperature of the fish approximated the temperature of the water within no longer than four minutes (see results of Experiment C) and since the primary coma often lasted as long as an hour, then i t must be that i t takes some time for the calcium and the other compounds affected to reassume the chemical 1 50. and physical combinations and distribution as existed originally in the unaffected c e l l . The low temperature of the cell with comcomittant low velocity of the reactions involved in restablishing the original equilibrium of the cell affected by chilling is probably at least partly responsible for this long delay in the return of "consciousness" to the fish since the coma=like state is considerably shortened i f the fish are placed in warmer water. To avoid confusion, one point should be emphasized for this phenomenon: the temperature at which the i n i t i a l shock and primary coma reactions occur are not necessarily related to acclimatization temperature; rather, it is the degree of change of temperature together with the rate of change that determine i f a fish exhibits these reactions. Finally, as noted in the Experiments and Results, the observation was made that the larger fish were slower to exhibit the ini t i a l shock and primary-coma reactions; i t is not improbable that this observed relationship is due to the less rapid rate of f a l l of the body temperature of the larger fish (see experiment C). In a similar experiment Colbert et al (10a) found that the rate of heat loss of alligators when placed in cold water was inversely proportional to the mass of the animal, being most rapid in the smallest individual and slowest in the largest alligator. Now i t has been shown that the ratio of surface area of a large animal body to its mass is less than the same ratio for a small animal body and that this fact, together with the effect of greater insulation of a larger animal body, is responsible for the slower transmission in the former of its body heat to the surrounding 51. environment. Even although small animals generally have a higher rate of heat production per gram of body weight than large animals (7), apparently the efficient cooling due to the larger surface area (relative to the weight) of the smaller animals more than offsets the heating effect of their higher metabolic rate. Thus i t is the differences in the rate of transmission of body heat to the environment that is responsible for the differences in the rate of f a l l of the body temperature of the goldfish,and therefore for the differences in the time of onset of their i n i t i a l shock reaction. As was noted in the introduction, Doudoroff (15, 16) has suggested that a major cause of the slow death of marine teleost fish by chilling is a failure of the body systems concerned with osmotic regulation (e.g. the integument, gill s , and the central nervous system). He further suggests that this failure results from the injurious action of the cold on the cells of these systems. According to the theory of Belehradek (4) as outlined in the Introduction, the injury and death of these cells would result from the increase in protoplasmic viscosity of the cells beyond a point compatible with l i f e . It is entirely probable that the cells of other parts of the body are also injured by the ch i l l temperatures but that in the case of the marine teleost the injury or death of the cells of the body systems involved in osmotic regulation is the first to be manifested because of the delicate balance involved in this type of regulation and because of the necessity of its continuance to prevent the cessation of l i f e . That the cells of other systems of the body are affected by the cold 5 2 . i s supported by Doudoroff fs (16) finding that Fundulus parviginnis lived longer than f i s h exposed in normal sea water but s t i l l died from the effect of c h i l l temperatures even though present in water approximately isosmotic with i t s body osmotic pressure. The death of the f i s h in this case would then be due to a disturbance of ome or more other functions within the f i s h . The slow death of goldfish by c h i l l temperatures i s not l i k e l y to be due to the exosmotic loss of water since i t is a fresh-water teleost. Thus, several authors (27, 29, U9, 50) have shown that the fresh-water teleost maintain their osmotic con-centration above that of the surrounding water. They do this by compensating for osmotic inflow of water by the active absorp-tion, presumably by the g i l l s , of salts from the fresh water and by the excretion of large amounts of a hypotonic urine. From Doudoroff*s (16) results using the marine teleosts Fundulus  parvipinnis and Atherinops, i t might be expected that the effect of the c h i l l temperatures on fresh-water f i s h would he to cause injury to the cells of the body organs and system concerned in the osmotic regulatory functions, as well as to the cells of other organs and systems. If such were the case then i t might also be expected that the balance of the osmotic equilibrium would be disrupted and consequently the osmotic inflow of water would not be compensated for. As a result, the f i s h would gain in water content and would die because of the resulting disruption of the balance between the water to salt and between the water 53. to protein content of the animal tissues2. That goldfish do gain in water content when exposed to near freezing temperatures has been shown recently by Platner (41a). Thus Platner found that gold-f i s h exposed for 60 hours to temperatures between 0.0°C, to 1.0°C. showed a decrease in hematocrit from 44.79 to 19.79 per cent. Although i t i s possible that similar results might not be obtained under the conditions of the experiments described heMn, these results of Platner do appear to support the hypothesis that death of goldfish by chillimg results from a disruption of the osmotic system. However, as he himself suggests, the possibility also exists that the hemodilution of Platner*s experiments may at least partially have resulted from the effect of excitement and sudden change of environment in causing the f i s h to swallow considerable quantities of water (l6b, 37a). Thus further experiments are necessary to show i f goldfish do gain in. water content under a l l conditions of exposure to cold and, i f an increase does occur, to show i f i t results from either or both a swallowing of water and a failure of the osmotic regulating system. One of the most important observations recorded in the experiments A and B was that, within the range of size comf pared, goldfish of the same or nearly the same age increase their tolerance to c h i l l temperatures as their size increases. 2. Smith (50) states that the variation i n the osmotic pressure per se i s not a detriment to the continuance of l i f e but that as a consequence of change in osmotic pressure the resulting change in water content with reference to the salt and especially to the protsin content of the tissues, i s a detriment to. l i f e . See also Krogh (29)* 5A. The most obvious explanation of this effect of size, especially when it is noted that some of the fish were up to three times the size of the others, would be that the larger fish have greater insulation than the smaller fish. The demonstration of a positive correlation between the survival time and the ratio of weight over length would seem to be in support of this supposition. With regard to the influence of insulation when a fish is placed into cold water, the results of experiment C show that there does exist a small difference in the rate of decline of the body temperature of fish under such conditions. It is not known i f this small difference of time effects even partially the differences in the survival times. From the results of experiments A and B where the fish are gradually cooled down so that there is l i t t l e or no body temperature difference between the fish of different sizes, i t would appear that the factor of insulation must be of importance during the comparatively long period of temperature equilibrium that occurs after the body temperature of the fish falls to or near that of the water. In addition, the fact that a positive correlation has been demonstrated between the survival times and the ratio of weight over surface area (i.e. equivalent to a negative correlation between survival times and the ratio of surface area over weight) would seem to indicate that the tolerance of the fish to chill temperatures is dependent also upon the ratio of surface area to weight with changing weight. Thus, as has been seen, it is the large fish with a smaller value for the ratio of area over weight that survives the longest. It would appear, therefore, that the factors of insulation and the decreasing ratio of surface area to weight for increase in size of fish would play some part in providing a greater tolerance to ch i l l temperatures. The major part that these two factors play in the tolerance of fish to chilling presumably would be their effect upon the rate ' of loss of metabolic heat to the surrounding water. Since it has been shown that because of the metabolic production of heat the temperature of fish can be slightly above that of surrounding water (19, 30), especially when acclimatized to the lower water temperatures (see the Introduction), then i t is not improbably,to suppose that small differences will exist in the body temperatures of different sizes of fish. Such small differences in body temperature would have a definite effect upon the survival times since, as has been shown by the work of previous authors, even the differences between a chill water temperature that will allow a fish to survive permanently and that which will cause its death is very small. Thus, for example, Fry et al (}8) have shown that for fish acclimatized to 24°C. approximately fifty six per cent of them will die i f they are exposed to 5°C. whereas approximately only fifteen per cent will die i f they are exposed to 4°G. From the above dis-cussion i t is conceivable that the two factors of insulation and the ratio of surface area to weight could influence the lengths of the survival times through their effect upon the rate of loss of metabolic heat and therefore upon the level of the body temperature. Although it might be expected from the results obtained 56. with goldfish, i t is not known i f the influence of increase of size of fish in increasing tolerance to chill temperatures exists in salt-water fish. In this respect, the principles just discussed in the previous paragraph for a fresh-water fish should apply also for a salt-water fish and therefore be responsible for an effect of size of the marine fish on their tolerance to chill temperatures. The resulting effect of these factors of insulation and ratio of surface area to weight can be tied in with Doudoroffs finding that osmotic disturbances are the cause of the slow chill-death of marine fish (16). This can be done i f it is postulated that the cells most vitally form affected by a disturbance of metabolic activity be those that ft»om the organs constituting the osmotic regulatory system. In relation to the disturbance of the proper functioning of the osmotic regulatory system itself, the observation of Keys- (27a) with Pacific K i l l i f i s h (Fundulus parvipinnis) would tend to point to a greater chill temperature tolerance of the larger fish; thus he pointed out that small fish have longer head length and therefore greater g i l l surface relative to body length. This greater relative g i l l surface of the smaller fish would !be to its disadvantage when exposed to chill temperatures with the consequent disruption of the osmotic regulatory systems that compensate for the exosmotic loss of water via the g i l l s , since a greater relative area is available for this loss of water. Also, i f the slow chill-death of fresh-water fish does involve a disruption of the osmotic regulatory system then these same factors should play a part in decreasing the tolerance of the smaller fresh-water fish. Thus, several factors (e.g. insulation, the changing 5 7 . ratio of surface area to weight with changing weight and g i l l surface area relative to mass, might conceivably be involved in the effect of size of fish on their tolerance to chill temperatures. In both fresh and salt-water fish i t is very probable that the compensatory increase in respiratory metabolism that occurs follow-ing the transfer of fish to low chill temperatures and which is mostly completed by the end of the first day, plays an important role in enabling the fish to increase its survival time or to resist slow death by chilling. That the rise in metabolic rate is considerable was mentioned in the Introduction where i t was noted that fish (gobies) kept at a low temperature and then placed at a common intermediate temperature with fish that had been kept at high temperatures had an oxygen consumption of almost twice that of the high temperature fish (56). It is not inconceivable, therefore, that this increase in the rate of metabolism could increase slightly the body temperature of the fish, thereby decreasing the protoplasmic viscosity. Such a reaction would give the fish a "breathing-spell", thereby enabling the fish to keep the balance between the vital systems of the body near enough to equilibrium to either increase' the length of the survival time or to maintain l i f e long enough to allow time for the slower acclimatization reaction of the transformation of the melting and solidification points of the protoplasmic fatty constituents to pro-ceed. The latter transformation reaction would decrease the viscosity of the affected cells s t i l l further. Thus, i f the above is correct, the rise in metabolic rate of fish following transfer to lower 5 3 . temperatures could be said to be the first aid to the fish in increasing its tolerance to chill temperatures whereas the decrease in the melting point and solidification point of its protoplasmic fatty constituents could be said to be the second slower acting aid. On the basis of this line of reasoning, one can, in theory at least, relate the temperature of death of a fish by slow chilling baok to its acclimati-zation temperature. It should be mentioned that the occurrence of the compensatory increase in the respiratory metabolism that occurs following the transfer of fish to chi l l temperatures is not entirely in agreement with the commonly held view of some authors and text-books (7, 5, 38) that in aquatic poikilothermio animals the metabolic rate is strictly proportional to the environmental temperature. In fact, this responding increase of the metabolism on exposure to the chill temperatures is remarkably like, i f not the same as, the chemical regulation of the horaoiotherm. This latter type of regulation, as noted in the Intro-duction, involves an increase in metabolic rate on exposure of the homoiothermic animal to very cold temperatures. Although Best and Taylor (7) have stated that fish do not show a chemical regulation, on the basis of these results i t can be conjectured that a chemical regulating system, controlled by the hypothalamus of the fish brain, exists in the fish also, but that because of the factors discussed in the Introduction (e.g., poor blood supply of the fish, large heat-absorbing capacity of water, and the lack of physical regulation of heatloss by fish) the increase in metabolic rate is not enough to 59. maintain a constant temperature of a fish in the face of a changing environmental temperature. In regard to this question, it is inter-esting to note that the chemical regulation against cold is commonly regarded as the more primitive type of regulation. While most of the theorizing of the present discussion is based upon experimental observations, related and unrelated to one another, i t is only too well realized that much more experimental work is necessary, especially with respect to the use of proper thermocouple technique (41) to measure and compare accurately the body temperature of fish of different size3, age, etc. under the various conditions of environmental temperature. Such work should be done in order to provide a firm basis of facts for the theerries in explanation of the phenomenon of injury and death of fish by slow and by rapid chilling. 6 0 . SUMMARY 1. A brief review of the mechanisms involved in and theories proposed to explain the injury and death of fish by exposure to chilling temperatures is presented. 2. Based upon the observation that the strength of electrical stimulus required to elicit a response from goldfish exposed to chilling temperatures increased until finally the fish would no longer respond, a method and an apparatus are described for determining more closely the true end-point of death of goldfish dying from this exposure to chill temperatures. 3. Detailed descriptions are presented of the " i n i t i a l shock", "primary chill-coma", and "secondary chill-eoma"# reactions as they were observed to occur in goldfish exposed to chilling temperatures. 4. Results are presented which show that the rate of f a l l of the body temperature of goldfish is inversely proportional to their size when taken from 70°F. and placed at 35.5°F. 5. Results show that, within the range of size compared, goldfish of the same or nearly the same age increase their tolerance to chill temperatures as their size increases. Statistical analysis of these results show a positive correlation between the weight of the fish over their length and the survival times as well as a negative correlation between the surface area of the fish over their weight and the survival times. 61 . 6. The results reported herein, as well as the related results of other investigators, are discussed and hypotheses are put forth in an attempt to explain the various phenomena associated with the injury and death of fish by slow and by rapid chilling. 62. REFERENCES » 1. Barbour, H.G. The Heat-Regulating MechaniBm of the Body. Physiol. Rev.,,, Is 295-326, 1921. 2. Bazett, H.C., Physiological Responses to Heat. Physiol. Rev., 7: 531-599, 1927. 3. Behre, Ellinor H., An Experimental Study of Acclimation to  Temperature in Planaria doretocphala. Biol. Bull., 35s 277, 1918 4. Belfthradek, J., Temperature and Living Matter. Protoplasms Monographien, 8:277 pp., Berlin, 1935, Gebruder Borntraeger. 5. 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