The Effect of Oxygen and Carbon dioxide 
 On the Development of Certain 
 Cold Blooded Vertebrates 
 
 By 
 
 Ada Roberta Hall 
 
 A. B. University of Oregon, 1917 
 A.M. University of Oregon, 1919 
 
 AY DIGEST-OP A THESIS 
 
 SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
 DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY IN THE 
 GRADUATE SCHOOL OF THE UNIVERSITY 
 
 OF ILLINOIS, 1921 
 
 Reprinted from Ecology Vol. V, No. 3 p. 290 and 
 Ecology Vol. VI, No. 2 p. 104 
 
Digitized by the Internet Archive 
 in 2022 with funding from 
 University of Illinois Urbana-Champaign 
 
 https://archive.org/details/effectofoxygenca0Ohall_0 
 
abt 
 
 [Reprinted from Eco.oey, Vol. V, No. 3, July, 1924.] 
 
 THE. EFFECT OF OXYGEN AND CARBON DIOXIDE ON THE 
 DEVELOPMENT OF EGGS OF THE TOAD, 
 BUFO AMERICANUS? 
 
 Apa R. Hat 
 
 University of Illinots 
 
 INTRODUCTION 
 
 The success of a species in a given habitat depends largely upon the part 
 which environment plays in the development of the individual, and the loca- 
 tion of the most sensitive periods in the life history. The toad, Bufo 
 americanus Le Conte, lives and breeds in warm, temporary, stagnant pools, 
 thick with suspended mud and decaying matter, subject to many fluctuations 
 in temperature and all other environmental factors, while the whitefish,” 
 must have clean cold water comparatively free from decaying materials, pre- 
 senting the minimum of daily fluctuations. This paper is an attempt to deter- 
 mine the effects of variations in dissolved gases, of a magnitude common in 
 the toad habitat, upon development, and to locate the sensitive stages in the 
 early life history. An index of sensitivity was also sought in the amount of 
 oxygen released from hydrogen peroxide. 
 
 MATERIALS AND METHODS 
 
 It was found that the toads at Urbana, Illinois, breed in a variety of 
 places from barnyard pools thick in manure and mud, to temporary pasture 
 puddles. A plentiful supply also breed in a small artificial lily pond on the 
 campus of the University of Illinois where the adults may be easily captured. 
 They begin laying early in the morning and will continue until noon or after 
 when brought into the laboratory. This affords a continuous supply of 
 freshly fertilized eggs throughout the morning, and is more satisfactory than 
 artificial insemination. A further advantage that these eggs possess for such 
 a study as this is the fact that the time from fertilization to the functioning 
 of the internal gills at 16° C. is very short, about twenty days. This makes it 
 possible to watch their growth from hour to hour and to see the effect of 
 changing the conditions. The eggs are large enough to see the stages well 
 under a hand lens. 
 
 The pH was determined by the use of Hynson, Westcott and Dunning 
 indicator sets with additional tubes for high and low values. The oxygen 
 determinations were made by the Winkler method. As the amounts of 
 
 Contribution from the Zoological Laboratories of the University of Illinois.244 
 2 Studied for comparison, results to be published later in FcoLocy. 
 
 290 
 
 No1833 
 
 ( 
 
291 ADA R. HALL Ecology, Vol. V, No. 3 
 
 B 
 it! ip - / 
 
 Fic. 1. Apparatus for measuring the oxygen content of small amounts of water by 
 the Winkler method. A, large tube 2.2 cm. diameter by 6.5 cm. length, capacity 26 cc., 
 total length from I to 2 is 17 cm. R to R heavy black rubber tubing enclosing glass 
 rings B C and D, diameter I cm. outside, .6 cm. inside. 
 
July, 1924 EFFECT OF OXYGEN ON THE TOAD 292 
 
 water in the experimental dishes were small, 200 to 400 cc., the use of 250 
 cc. bottles was impracticable. A piece of apparatus was therefore devised 
 that would handle small amounts of water (Fig. 1). By raising and lowering 
 the mercury cup, water was siphoned into the apparatus from the experi- 
 mental dish, and the cocks 1 and 2 closed. B was filled with manganous 
 chloride (sol. I, Winkler method), C with potassium hydroxide-iodide (sol. 
 IT), and D with concentrated hydrochloric acid, and clamps 3, 4, and 5 
 closed after each addition. The clamps were then removed one at a time in 
 the same order and the liquids mixed by means of gravity, and a glass bead 
 inserted with solution I. The 26 cc. of water which the apparatus holds was 
 then titrated against N/4o sodium thiosulphate and the oxygen in cc. per 
 liter calculated. 
 . EXPERIMENTAL DATA 
 
 A. Physiological Life History from Fertilization to Internal Gill Stage 
 
 In this work on physiological life history several types of experiments 
 were performed: (1) Keeping the pH constant (8.0) the oxygen content 
 was varied, four concentrations, .4 cc., .9 cc., 1.41 cc. and 4.64 cc. per liter 
 being used. (2) Keeping the oxygen about the same in all dishes, .4 cc. to 
 .8 cc. per liter, the pH was varied from 6.1 to 8.0. (3) Eggs were sealed into 
 
 abcd ¢€ fghi jy k om n ° Paodtes t BDicds Cel teh) Tal} wen n S ahs 1s 
 
 1. I 
 
 Fic. 2. Toad eggs put into experimental conditions: I at 1 cell stage, II 2 cell stage, 
 and III 4 cell stage. 
 
 a fertilization, b 2 cell, c 12-16 cell, d early blastula, e late blastula, f blastopore lip, 
 g yolk plug large, h yolk plug medium, i yolk plug small, j early neural folds, k late 
 neural folds, m elongation, n tail differentiating, o tail 1/4 body, p tail 1/3 body, r tail 
 1/2 body, s tail 2/3 body, t tail equal to the body (7 days), u 8 days, v 9 days, x point 
 of death. 
 
293 ADA R. HALL Ecology, Vol. V, No. 3 
 
 known quantities of water with differing oxygen content and the time and 
 extent of development noted. 
 
 Time for normal development of toad eggs at 16° C., plotted as a 
 straight line in graphs of figs. 2 and 3, is as follows: Fertilization o hrs.; 
 first cleavage (2 cell), 3 hrs. and 20 min.; 4 cell, 4 hrs. and 30 min.; 8 cell, 
 6 hrs.; 12 to 16 cell, 8 hrs.; early blastula, 10 hrs. and 30 min.; late blastula, 
 23 hrs. and 30 min.; blastopore lip, 28 hrs.; yolk plug-large, 31 hrs: and 30 
 min.; yolk plug medium, 41 hrs.; yolk plug small, 48 hrs.; early neural folds, 
 51 hrs.; late neural folds, 62 hrs.; elongation, 72 hrs.; out of jelly—tail dif- 
 ferentiating, 95 hrs.; tail 1/4 length of body, 114 hrs.; tail 1/3 length of 
 body, 123 hrs.; tail 2/3 length of body, 143 hrs.; tail equal to body, 168 hrs.; 
 gills three, 192 hrs. — 
 
 TasLe I. Time in hours for reaching various stages of development in 
 the toad experiments 
 
 Read down the columns; embryos died where the number is underscored. 
 
 Series I. Put in 25 Min. Series II. Put in at 
 before Ist Cleavage 2 Cell Stage 
 A-1 | A-4 | C1 C-2 A-1I A- GI C-2 
 1 Sythe 3 eects eae 8.0] 8.0 | 5.8-6.6 | 6.0-6.4 8.0 8.0 |5.8-6.6 | 6.0-6.4 
 Oxygen Content (c.c.).. 4| 4.64 8 4 4 4.64 8 4 
 Time from fert. to: 
 TStACleaVage ee es eis 2254s. 3.5 Be 5 3.5 3.5 25 3.5 
 Farly ‘blastula ie sacs See BAe eats Mee | utiee de @ Ieee are: Nate 24 24 
 Late blastula........ 24 56 24 24 24 24 (Sate Ae SO 
 Blastopore lip....... on Woah ode 56 56 60 5On) § | Senet leeeem sere 
 Wolk plug: larve (eee sock), ey ed eee ot oe ee ene 734 ll, Sage sce Seer 73 
 Wolk plug medians enogi ss... s| ease FB 0 | Sisiete! choos | Saetere cea chee ee | peers rete ae Haas 
 May neural foliage. | o..-< oem hee Pte 7 3 te eee hs RS eo ath pol is conse 
 ote neural folds act 73. |ncen eee 95 95 Zien Reeds PR Cis abies” 
 Blonyation 15ee6. eee tie Tew led nee 123 123 Oot, doen eam 
 ail iditter peers 95 else Aletsic is So cal Se od py) Mian Wilbon sea aso. 
 Jail 4 body: the roc} 123 “YI23 | case ae ee Prodi awehS tale 2 Se alle doers: 
 Maileyasbodyacrencroe 1 be Mega eee Pept Mie UE A deka te ce |i il Read UAONET cto htatod. coco 
 Tail ibody..ea.4: TOSP ph TAS Ti aega el cle iene oc eet LOB Me. eae ee atioe Lae 
 Taylt24 ibody aaarnac LOZ) | LOB. ei cee APA eer 5 ees reiesetel cliche ete | ceca cts gee 
 Cail s=sbody oi aise s als Fea 64 LO QUI fd nate aie ole a ate heptiene wher sllhe 2s inc eee 
 
 In the first type of experiment eggs were put into the experimental dishes 
 at different stages of development, and the effects as shown by retardation, 
 acceleration or death noted. These results were tabulated in Table I, while 
 the A series and the same results are shown graphically in figures 2 and 3. 
 In making the graphs, normal development was taken arbitrarily as a straight 
 line from fertilization at o hours to the internal gill stage at 196 hours. 
 Time in hours was plotted as ordinates, and the stages of development, or 
 abscissae, were obtained by dropping perpendiculars from the normal de- 
 velopment line to the + axis according to the number of hours required to 
 
July, 1924 EFFECT OF OXYGEN ON THE TOAD 294 
 
 reach that development. All experimental curves were then drawn with both 
 time and period of development fixed by the normal curve. When the ex- 
 perimental curve rises above the normal line retardation is indicated; when 
 it drops toward the normal or below it, acceleration is indicated. Curves 
 parallel to the normal denote normal rate of growth. 
 
 = 
 
 ab cd Cn tig hell a m n ° Pp 
 0 F 
 
 Fic. 3. Toad eggs put into experimental conditions: I at late blastula, II at yolk 
 plug, and III at blastopore lip stages. Notation same as Fig. 2. 
 
 Using a pH of 8.0 and four concentrations of oxygen, .4 cc., .9 cc., 1.41 
 cc., and 4.64 cc. per liter, we find that a certain degree of development was 
 attained regardless of the concentration, death not occurring in any of the 
 experiments short of the yolk plug stage even when the oxygen was as low 
 as .4 cc. per liter. Eggs put in before the first cleavage (Fig. 2-I) show 
 marked retardation up to the late blastula stage then some acceleration so 
 that further development approaches normal. Eggs exposed to the experi- 
 mental conditions between the first cleavage and the late blastula stage (Figs. 
 2 and 3) show a period of eighteen or twenty hours with little or no retarda- 
 tion and then a period of quite marked retardation occurring in the blastopore 
 or yolk plug stages. Later development for the higher concentrations of 
 oxygen again approaches normal. Eggs put in after the late blastula stage 
 (Fig. 3) show general retardation for the lower oxygen concentrations but 
 fairly normal development for the higher concentrations. 
 
 The results of this type of experiments show that where the oxygen was 
 as low as .4 cc. to .9 cc. per liter death occurred in part of the experiments, 
 but at higher concentrations only retardation showed the effects of exposure. 
 At the late blastula and blastopore lip stages the higher concentrations even 
 caused acceleration of growth. The most sensitive stages appeared to be 
 from fertilization to the first cleavage, and the gastrulation stages. 
 
295 ADA R. HALL Ecology, Vol. V, No. 3 
 
 _ The second type of experiment was where the oxygen was low (.4 to 8 
 cc. per liter) and the pH varied, 6.1, 6.4, and 7.0 to 8.0. The stages reached 
 in development are shown in the graphs of Figs. 2 and 3. Exposures to these 
 conditions beginning before the four cell stage gave death with retardation 
 in 24 to 120 hours, c to k. The same effect is seen in eggs put in after the 
 eight cell stage. Those exposed between the four and eight cell stages show 
 the greatest development (198 hrs.) which is past the sensitive stages. With 
 small amounts of oxygen the effect was much more rapid and deadly with a 
 pH of 6.1 to 6.4 than with a pH of 6.4 to 6.8. 
 
 In the third type of experiment a dozen eggs were placed in an eight 
 dram vial (about 26 cc. of water) supplied with a two-holed stopper having 
 the inlet tube extended to the bottom of the vial and the oulet tube even 
 with the stopper. Both tubes ended outside the stopper in short rubber 
 tubes. Water of the desired oxygen content was run through the bottle 
 until all oxygen due to air was flushed out and then a clamp on the two rubber 
 tubes sealed the bottle. Development was watched by means of a dissecting 
 lens until growth stopped. The oxygen content was then determined by 
 adding the chemicals through the inlet tube, being careful to avoid air bubbles 
 when inserting the pipettes in the rubber tubes. The cleavage of a dozen eggs 
 proceeded to the late blastula stage regardless of the period of development 
 when put in or the amount of oxygen present (.1-5.7 cc. per liter). Further 
 development was roughly proportional to the amount of oxygen present, as 
 was also the time to death. 
 
 B. Reaction of Eggs to Hydrogen Peroxide as Development Progresses 
 
 A universal property of protoplasm (both plants and animals) is the 
 abitilty to liberate oxygen from hydrogen peroxide. What this property is 
 due to is a debated question at present, but in general it is ascribed to an 
 enzyme called catalase. Several theories have been advanced as to its sig- 
 nificance in animal life. References are given to these in the literature 
 cited (Becht ’19, Burge and Burge ’21, Zieger ’15). As a measure of the 
 sensitivity of the developing egg at different stages in the early life cycle the 
 reactions to oxygen and carbon dioxide (acidity) have been discussed. It 
 was thought that a study of the amounts of oxygen released from hydrogen 
 peroxide at various periods of development might be of significance. Ac- 
 cordingly a series of determinations was made with the toad egg using the 
 method outlined by Burge (16), Fig. 4A. A second series was made for the 
 toad eggs the following spring using eight dozen eggs instead of one dozen 
 and shaking the material by machine instead of by hand (Fig. 4B). A com- 
 parison of A and B shows a striking parallelism and the two curves seem to 
 confirm each other. 
 
 From a study of these curves (Fig. 4) it may be seen that there was a de- 
 crease in the power of liberating oxygen from hydrogen peroxide from the 
 
July, 1924 EFFECT OF OXYGEN ON THE TOAD 296 
 
 unfertilized egg to the fertilized, an increase from fertilization to the early 
 blastula, a decrease from early blastula to elongation of the embryo, and from 
 this point to twenty-one days a steady increase. The stages most sensitive 
 to low oxygen and high acidity were (1) fertilization to first cleavage, and 
 (2) the gastrulation stages. These were the low points on this curve. Is 
 there a correlation between sensitivity and a lowered power of liberating 
 oxygen from hydrogen peroxide? 
 | 
 
 | 
 cc icslonsed by 
 
 200(10 
 18049 
 160}8 
 
 10075 | ae 
 : Samat eel 
 
 8074 | pe sag ec | 
 
 6073 - 
 
 ov ab cd Sur ehh ey key im n ° p r s t u v 
 
 Fic. 4. Amounts of oxygen released from hydrogen peroxide at different stages of 
 development of toad eggs. Series A determinations were made with one dozen eggs in 
 1920. Series B was made using eight dozen individuals in 1921. 
 
 Winternitz and Rogers (’10) have shown a definite increase in what they 
 call catalase for the different stages of the hen’s egg, as development pro- 
 ceeds. Burge and Burge (’21) have shown an increase in this power from 
 egg to adult for the Colorado potato beetle. Zieger (’15) has found a definite 
 rythm for catalase in the insect life history, reporting that the power is high 
 where rapid growth or metamorphosis is going on (early larval stages, 
 pupal stages) and low during resting stages. Child in his book “ Senescence 
 and Rejuvenescence’”’ has shown that metabolism is high during the more 
 sensitive stages. Burge believes that increase in catalase brings about an 
 increase in metabolism. This curve for the toad egg seems to be a contradic- 
 tion of one statement or the other since it shows the power of splitting 
 oxygen from hydrogen peroxide to be low at the sensitive stages. More 
 work is needed on complete life histories before definite conclusions can be 
 drawn about this point. 
 
 I wish to thank Dr. V. E. Shelford, at whose suggestion this work was 
 undertaken, for his helpful advice and kindly criticism during the course of 
 the investigation. I am indebted to Dr. W. E. Burge and Dr. H. B. Lewis 
 for suggestions in the enzyme work. 
 
297 ADA R. HALL 
 
 SUMMARY AND CONCLUSIONS 
 
 1. Conditions of high carbon dioxide or low oxygen content such as may 
 occur in the normal environment and which are tolerated by the other periods 
 of development, cause great retardation and even death at sensitive stages. 
 The effects of low oxygen are much more detrimental in acid than in alkaline 
 water. 
 
 2. There are two definitely sensitive stages in the early physiological life 
 history of the toad (a) one cell stage; fertilization to first cleavage, (b) the 
 gastrulation stages; blastopore lip formation to the small yolk plug stage. 
 
 3. There is a decrease in the amount of oxygen released from hydrogen 
 peroxide at the sensitive periods which roughly corresponds in magnitude to 
 the decreased resistance. 
 
 LITERATURE CITED 
 
 Becht, F. C. 1919. Observations on the Catalytic Power of Blood and Solid Tissue. 
 Am. Jour. Physiol., 48: 171-1901. 
 
 Burge, W. E. 1016. Relation between the Amount of Catalase in the Different Muscles 
 of the Body and the Amount of Work Done by these Muscles. Amer. Jour. 
 Physiol., 41: 153-161. 
 
 Burge, W. E., and Burge, E. L. 1921. An Explanation for the Variation in the In- 
 tensity of Oxidation in the Life Cycle. Jour. Exp. Zool., 32: 203-206. 
 
 Coventry, A. F. 1011. Note on the Effect of Hydrochloric Acid, Acetic Acid, and 
 Sodium Hydroxide on the Variability of the Tadpole of the Toad. Arch. Entw. 
 Mech., Band 31: 338-341. 
 
 Hall, A. R. 1918. Some Experiments on the Resistance of Sea-urchin Eggs to Sul- . 
 phurous Acid. Pub. Pug. Sd. Biol. Sta., 2: 113-1109. 
 
 Jenkinson, J. W. 1010. The Effect of Sodium Chloride on the Growth and Variability 
 of the Tadpole of the Frog. Arch. Entw. Mech., Band 30: 349-356. 
 
 Winternitz, M. C., and Rogers, W. B. t1g10. The Catalytic Activity of the Developing 
 Hen’s Egg. Jour. Exp. Med., 12: 12-18. 
 
 Zieger, Rudolph. 1915. Zur Kenntnis der Katalase der neiderer Tiere. Biochem. 
 Zeitschrift, 69: 39-110. 
 
{Reprinted from Ecoxocy, Vol. VI, No. 2, April, 1925.] 
 
 EFFECTS OF OXYGEN AND CARBON DIOXIDE ON THE 
 DEVELOPMENT OF THE WHITEFISH? 
 
 Apa R. Hatt 
 University of Illinois 
 
 Introduction 
 
 A subject of vital interest is the rhythm of events in the physiological life 
 history of any species, and the manner in which this rhythm may be affected 
 by environmental factors. Accordingly this work has been undertaken for 
 the purpose of: (1) finding out the relative sensitivity of the stages in the 
 early life history of the Whitefish; (2) testing the resistance and reactions 
 of normally hatched individuals as compared with the reactions of those 
 hatched under experimental conditions. 
 
 Materials and Methods 
 
 The material for this work was the lake whitefish, Coregonus clupeiformis 
 Mitchill. Some work was done on fertilization and early cleavage stages at 
 the U. S. Hatchery, Put-in-Bay, Ohio. The major part of the work, how- 
 ever, was done at the Vivarium, University of Illinois, on material shipped 
 from the hatchery in ice. The temperature of the lake water was about 8° 
 C. at the beginning of the season. It had a pH of 7.0 and an oxygen content 
 of 4.08 cc. per liter. At the Vivarium the stock was kept in water from the 
 University wells aerated to about 2.6-3.3 cc. per liter, and with a pH of 7.8. 
 It was cooled to 10° C. by means of brine coils. The water (pH 8.0-9.0; 
 carbonates 232 + parts per million) for all the experiments was boiled free 
 of all dissolved gases, and part of the salts precipitated (Shelford ’18). 
 
 The apparatus used for varying the pH and oxygen content is shown in 
 figure I. Bottle 1 contained approximately N/4 sulphuric acid which 
 siphoned over into the mixing bottle 4. The stopcock 2 controlled the flow 
 which could be measured by counting the drops through the glass bulb 3. 
 Thus a known amount of the acid was added to a known flow of the boiled 
 water which entered at W. This water was also 10° C. The water leaving 4 
 had a known pH, and was oxygen free. Since it contained carbonates, acids 
 set free carbon dioxide which changed the pH. The oxygen was controlled 
 by adding compressed air (see fig. 1 and explanation). Three such sets of 
 apparatus were used giving three hydrogen ion concentrations, each with 
 three oxygen concentrations. 
 
 1 Contributions from the Zoological Laboratory of the University of Illinois, No. 256. 
 
 104 
 
105 ADA R. HALL Ecology, Vol. VI, No. 2 
 
 The pH was determined by the use of Hynson, Westcott, and Dunning 
 indicator sets with additional tubes for high and low values (Clark ’20). 
 Brom cresol purple (5.8-6.6), brom thymol blue (6.6—7.6) phenol red (6.6— 
 8.0), and thymol blue, alkaline range (8.0-9.2) were used. The colorimetric 
 
 nee 
 
 alias 
 
 Fic. 1. Apparatus for the control of experimental conditions. Method of varying 
 hydrogen-ion concentration and oxygen content. 1, N/4 sulphuric acid; 2, cock for con- 
 trol of acid by drops at 3; 4, mixing bottle for acid and boiled water from W; 5, 6, and 
 7, jars, to which air from A is added in varying amounts; C, cocks for control of air 
 flow; 8, 9, and 10, half-pint sedimentation glasses for eggs; M, mercury manometer for 
 keeping air flow constant. 
 
 standards were checked electrometrically by Dr. R. E. Greenfield. Oxygen 
 determinations were made by the Winkler method with an apparatus which 
 used only 26 cc. of water for a determination (Hall ’23). 
 
 Experimental Data 
 EXPERIMENTS AT THE HATCHERY 
 
 All the experimental work was done at 10° to 11° C. at the U. S. Hatch- 
 ery. The length of life of eggs and sperm was tested. Three sets were run 
 (a) dry eggs and sperm, (b) dry eggs and sperm mixed thoroughly with 
 water to uniform milky fluid, and (c) dry sperm and eggs standing in lake 
 water. With wet eggs and dry sperm eight minutes was the latest time at 
 which fertilization was possible. With wet sperm and dry eggs nine minutes 
 showed a small number of fertile eggs. But with both eggs and sperm dry 
 fertilization took place at seven and a half hours. It may have been possible 
 after longer time as such an experiment was destroyed and could not be veri- 
 fied. 
 
April, 1925 DEVELOPMENT OF THE WHITEFISH 106 
 
 In early development the eggs were exposed to constant oxygen (4.08 cc. 
 per liter) but with varying pH. Normal lake water had a pH of 7.0. 
 Acidity (pH 6.2-6.6) was produced by adding sulphuric acid, and alkalinity 
 (pH 8.4-8.6) by adding sodium hydroxide. Dishes of standing water were 
 used and changed frequently. Eggs fertilized directly in the solutions were 
 normal at the end of twenty-four hours (32-64 cell stage). This experiment 
 was then repeated using lake water boiled until the oxygen content was 2.9 
 cc. per liter. Fertilization and development occurred in all the dishes, but 
 there was a marked difference between the acid and alkaline waters. With 
 a pH of 6.2 and 6.6, respectively, 80 per cent and 25 per cent fertilized and 
 developed; with a pH of 7.0 only 3 per cent, and at a pH of 8.4 but I per 
 cent. A hydrogen ion concentration which is too great to favor later de- 
 velopment appears best for fertilization (Cohn 718). 
 
 EXPERIMENTS WITH SHIPPED Eccs 
 
 In the work on later stages with treated running water at the University 
 of Illinois, special attention was paid: (1) to keeping conditions as near 
 constant as possible from day to day; (2) to watching the stages of develop- 
 ment reached in each concentration and comparing these with each other and 
 the control stock; (3) to working out the death rate and the percentage 
 hatching for each concentration; and (4) to testing the vitality and reactions 
 of the larvae hatching from the different stocks. 
 
 In this work apparatus was set up in triplicate (fig. 1). Values of pH 
 6.4, 7.0, and 8.0-9.0 respectively were used, each pH with an oxygen content 
 of I cc., 3 cc., and 4.5 cc. per liter; pH was taken in all the dishes each day, 
 and oxygen was determined every two or three days. 
 
 The eggs were obtained from the hatchery in four lots. The first lot, 
 spawned December 2, was in the thirty-two cell to early germinal cap stage 
 when received December 3. The second lot, spawned December 5 and re- 
 ceived December 24, had the tail just elongating and the fin buds starting. 
 The eye vesicles had formed but no pigmentation had occurred. The third 
 lot, spawned December 7 and received January 31, were only slightly far- 
 ther along in development than lot two. They had just begun to show the 
 pigment in the eyes. The fourth lot, recetved March 12, were fully devel- 
 oped and started to hatch immediately. 
 
 A comparison of the chemical analyses of Lake Erie water (the average 
 of Huron at Port Huron and Erie at Buffalo, Clarke ’20, p. 70) and the 
 boiled University water is important in connection with the experimental re- 
 sults obtained with the whitefish, and is given in Table I. 
 
 The four stocks differed in: (a) the length of time in these two kinds of 
 water, (b) temperature at hatchery and in experiments, and (c) the stage at 
 which shipment was made. 
 
107 ADA R. HALL Ecology, Vol. VI, No. 2 
 
 Tas_e I. Comparison of Lake Erie and boiled University of Illinois water 
 
 Lake Erie Water Boiled U. I. Well Water 
 
 DE (COs) paket Beet ere 7.0 9.0 
 
 GOr 5 Beka Sone ek bee ee 54 ppm. 250 ppm. 
 
 SOG Say foe Rahn ae che eee pee 10 ppm. trace 
 
 Oy MPA ao et att tat char hs fy Shite 5 ppm. I ppm. 
 
 Cas Sher Sah een rs 27 ppm. 33 ppm. 
 
 IMS aachote wicteca cee Ie ae tee ee 7 ppm. 27 ppm. 
 Nasik oc) ie esi ce ce eee ee eee 5 ppm. 16 ppm. 
 ‘Lotal sods a, oo ttn, ve aoe eee 112 ppm. 248 ppm. 
 
 TABLE II. Showing the development of whitefish eggs under different conditions 
 
 Figures are days to stage indicated on left. Dates of spawning, receipt at Urbana, 
 and beginning of experiment, respectively, follow each series number. Series II and 
 III are stock 1; series VIII is stock 3. The entries in italics indicate that embryos died 
 soon afterward. Oxygen all given in cc. per liter. 
 
 A-I B-1 C-1 A-2 B-2 C-2 A-3 B-3 
 Series II; 12/2; 
 T2/39 12/6: 
 DIderan Se emer meee 6.2-7.5| 5.8-8.9| 6.4-8.1| 6.4-8.0] 6.2-8.9] 7.9-8.5| 6.6-8.3| 6.8-9.2 
 pHimea nine ges vee: 6.2 6.2 6.4 7.0 We We Fest » Oh ENG 
 Oxygen range....... o—.9 0-1.04 | 0-2.08 | 0-.9 0-1.24 | .0I-3.7] 0-.9 O-1.7 
 Oxygen average..... cD 2 6 is LZ a7. gi 3 
 Cap small cells...... 4 4 4 4 4 4 4 4 
 Embryo forming. ...| 5 5 5 5 5 5 5 5 
 Post ring large...... 6 6 6 6 
 Post ring small...... 7 7 7 ii 7 7 7 10 
 Eye vesicle......... 
 Pailtilat. fee see Io 10 9.5 IO 10 9 10 10.8 
 ailestartin awe see 12 II rt 10 II 
 Tail elongating...... 13 1g 13 II 
 Tails, Doadye «:. eae 13 
 Series III; 12/2; 
 fofss 12/17, 
 pHiiangers! ..cieere 5.9-6.9| 6.8-7.0] 6.8-9.0 cae 6.8- 7.0] 6.8-9.0 Be 6.8-7.0 
 DHtMednt eee 6.4 8.5 6.4 8.5 6.4 
 Oxygen range....... .4-2.4| .4-2.2] .2-1.7] 1.3-3.1| 1.9-3.7| I.7-3.2] 3.2-5.3] 3-3-4.9 
 O: © ~eniaverage: ..... 1.64 .88 1.07 2.32 2.56 2.49 4.37 3.96 
 i ‘igmented...... 15 15 15 15 15 15 15 15 
 Fitrays!short. .2--.|19 19 7 IQ 17 17 19 17 
 Fin rays 4 fin...... 21 21 21 
 Tail near head...... 26-27. |20 26 26 26 
 ‘Dail totheadiver-es 27 27 27. 2 
 Wail-toveyene erm 
 Hatching<feeee es. 
 Series. VIII; 12/7; 
 D202) oy 
 pH rangesaee ee eee 6.1-7.2|. 6.2-9.0| 9.0 6.1-7.2| 6.2-9.0] 9.0 6.1-7.2| 6.2-9.0 
 DH meanest ce 6.4 eS 9.0 6.4 7.6 9.0 6.4 7.6 
 Oxygen range....... OV siete Vanes 18—2°4/02,0—3°2|\ aa —oe5 2 0-30] asd oko eres 
 Oxygen average..... 61 4 1.6 2.6 2.12 3.0 3.5 3.8 
 Ready to hatch..... 76 76 76 76 76 76 76 76 
 Hatching—tst...... 78, 7% |78, 13%|78, 18%|78, 18%|78, 16%|78, 15 %|78, 8% |78, 17% 
 
 Max. hatching...... 79-82 |79-82 |79 81-83 |79-82 |79-81 {81-83 {81-83 
 
April, 1925 DEVELOPMENT OF THE WHITEFISH 108 
 
 Effect of Conditions on the Different Stocks 
 
 Table II shows the number of days required to reach the different stages 
 in the various concentrations. The numbers and letters used below will be 
 found in this table. 
 
 Stock r. In series II, conditions varied materially in all dishes. With 
 a pH around 6.2-6.4 we find that a set of eggs put into the dishes 4, B and 
 C-1 on the seventh day varied in both amount of development and length of 
 life roughly in proportion to the amount of oxygen supplied. This was 
 I ce., .2 cc., .6 cc. per liter respectively. This amount was not enough to 
 carry development beyond the elongation of the tail bud, and was lethal in 
 every case. This same oxygen effect seemed to hold with higher pH, and 
 was evidently below the threshold of whitefish development. 
 
 In series III, oxygen content was raised so that dishes No. 1 had bagel 
 I cc. per liter, dishes No. 2 about 2.5 cc., and dishes No. 3 about 4 cc. per 
 liter. With this increase in oxygen concentration the more alkaline water 
 showed the farthest development. The eggs lived as long in the acid and 
 neutral waters of high and low oxygen but development was retarded. It 
 is significant that those eggs in neutral waters were more retarded than those 
 in either the acid or alkaline waters. Where neutrality was maintained as 
 in this set a detrimental effect is noticed in development equal to that of 
 higher hydrogen ion concentrations. The results suggest that pH of 7.8 is 
 optimum and 7.0 too high a concentration of hydrogen ions. This lot of 
 eggs, put in at the early pigmentation stage of the eyes lived nearly to hatch- 
 ing. 
 
 Stock 2. The experiments of series IV and V were started three days 
 apart and run simultaneously so that conditions were identical except for the 
 time of entering the experimental waters. The average oxygen for the 
 different dishes was slightly higher than in series III. The eggs of series 
 IV were put in when the fin buds were just starting. A distinct oxygen dif- 
 ferential was established: the low oxygen eggs developed to the point where 
 the tail reached the mendian line of the head; the medium oxygen eggs to the 
 point where the tail reached past the head and around to the eye; while the 
 high oxygen eggs developed just to hatching in the acid and neutral waters. 
 The alkaline water did not seem enough lower in oxygen to account for the 
 retardation which occurred, and this retardation was probably due to the 
 hydrogen ion concentration. 
 
 In series V more difference in development is apparent between the dif- 
 ferent dishes. The greatest amount of retardation occurred in the low 
 oxygen jars and in the high oxygen of the neutral series (pH fluctuating). 
 Two eggs hatched in the medium oxygen of the neutral series, but all 
 others died just before hatching time. These were the first individuals to 
 
109 ADA R. HALL Ecology, Vol. VI, No. 2 
 
 hatch in the experimental dishes, though a few hatched in the stock (pH 7.8 
 and oxygen 3.3 cc. per liter). 
 
 Stock 3. Hatching occurred in the experiments run with both the third 
 and fourth stocks. As these stocks were older, the time of exposure to the 
 experimental waters was later, and a different reaction resulted. Series VI 
 and VII were started two days apart but were run simultaneously. Hatch- 
 ing occurred in several of the dishes and extended over several days, the 
 beginning and duration of the process being recorded in Table II. The per 
 cent hatching is given as the second member opposite ‘“‘ Hatching-Ist.” 
 
 Series VIII was started just as the eggs of stock 3 were ready to hatch, 
 to test the effect of different concentrations on this process and on the life of 
 the larvae. Hatching occurred in all the dishes beginning on the same day. 
 Maximum hatching was 2 days later in the acid “medium” oxygen and 
 in the acid and neutral high oxygen, showing some retardation for these con- 
 centrations. 
 
 Stock 4. Series IX is the result of the exposure of eggs reared for the 
 entire time of development at the hatchery in lake water. It shows the 
 relative sensitivity of the hatching period and the newly emerged larvae. 
 The first hatching occurred in the acid and neutral low oxygen an hour or 
 two after putting the eggs in the water. A few also hatched in alkaline 
 “low” and “ medium” oxygen later the same day. 
 
 Differences in the Stocks at Hatching 
 
 The normal time for the development of these eggs at the hatcheries was 
 four to five months. Due to higher temperature my first stock began hatch- 
 ing at thirty-two days. The newly hatched larvae from each of my four 
 stocks were measured for body length, and for size and shape of yolk, and 
 the time from hatching to death recorded. 
 
 Only a few individuals were measured in stocks I and 2. In stock 1 the 
 fry were 8 to 11 mm. in length with round yolk sacs 2 mm. in diameter. 
 These lived only two days. In stock 2 the fry were 11 mm. long, also with 
 round yolk sacs. These lived sixteen days with no food. Stock 3 averaged 
 II.I mm. with similar yolks and lived at least ten days. Stock 4, raised in 
 lake water, gave fry of a distinctly different type. They were 13.4 mm. to 
 15 mm. long and were extremely active. The yolk sacs were oval but of the 
 same volume as the earlier stocks. These lived for twenty-five days without 
 food, and at the time of death measured 15 mm. This shows that eggs reared 
 for a long period at low temperature attain a larger size and have more 
 energy than they would have if forced at higher temperature (an argument 
 against forcing conditions in hatchery work). 
 
April, 1925 DEVELOPMENT OF THE WHITEFISH 110 
 
 Mortality 
 
 Due to Fungus. One factor in mortality was fungus growth. This was 
 in direct proportion to the amount of oxygen present, therefore was most 
 detrimental in the high oxygen concentrations. The pH seemed to have no 
 differential effect. The fungus grew very rapdily on dead eggs but also en- 
 tangled live ones and killed them. Infected eggs left in the dishes would 
 mat dozens of eggs together and kill them in a day or two. Since higher 
 temperatures were more favorable to the fungus it grew more abundantly 
 in my dishes than at the hatchery. 
 
 Due to Experimental Conditions. The eggs of the different series (II to 
 IX) were counted and the dead recorded from day to day. From this data 
 the per cent of deaths was calculated for each dish of the series. The stages 
 from early formation of the embryo and ring seemed very sensitive, as in all 
 concentrations the deaths quickly amounted to 100 per cent. From the time 
 of closure of the posterior ring and beginning of elongation of the tail and 
 fin buds sensitivity decreased, as shown by the following figures: 
 
 Series I]: Early gastrulation. At the end of three days the per cent which 
 had died was as follows: A-1, 100 per cent; B-1 100 per cent; C-1, 66 
 per cent; A=2) 100 per cent; B-=2, 75 per cent; C-2, 50 per cent; A-3, 
 100 per cent; B-3, 95 per cent; C-3, 50 per cent. 
 
 Series III: Eye pigmented, lens showing. At the end of nine days the per 
 cent which had died was as follows: A-1, 20 per cent; B-1, 20 per cent; 
 (=I, 10° per cent; A=2, 50: per cent; B-2/ 100 per, cent; C-2; 5° per 
 cent; A-3, 10 per cent; B-3, 50 per cent; C-3, 5 per cent. 
 
 Series IV: Tail 1/4 body length. At end of sixteen days the per cent which 
 had died was as follows: A-1I, 91 per cent; B-1, 26 per cent; C-1, 49 
 per cent; A-2, 64 per cent; B-2, 96 per cent; C-2, 98 per cent; A-3, 
 70 per cent; B-3, 66 per cent; C-3, 98 per cent. 
 
 The deaths due to fungus were in most cases small compared to total deaths. 
 Total mortality is the index to the effect of varying conditions of oxygen and 
 carbon dioxide, and shows the relative sensitivity of the different ages of 
 eggs. 
 
 Effect of Other Acids 
 
 As sulphuric acid liberated the carbon dioxide in the above experiments, 
 it was thought desirable to run a check with other acids. A series of finger 
 bowls was prepared such that a pH of 6.3, 7.0, and 9.0 was obtained for each, 
 sulphuric, hydrochloric, and acetic. Salts such as sodium chloride an- 
 tagonize the acid which may be present in water (Loeb, ’12, Osterhout, 714), 
 and therefore a duplicate series was made up in which one third of the volume 
 of water was substituted by M/6 sodium chloride. The same amount of 
 
III ADA R. HALL Ecology, Vol. VI, No. 2 
 
 mixing was given each solution as it was made up to insure a uniform oxygen 
 content of 1.5 cc. per liter. The finger bowls were filled full and covered. 
 Readings were taken without uncovering. Table III shows the time to death 
 in the different concentrations. In the acetic and sulphuric acid (no salt) 
 death occurred in pH 7.0 first, 9.0 second and 6.3 last, but for hydrochloric, 
 death in 6.3 preceded 7.0. Neutrality was most quickly fatal, and alkalinity 
 more toxic than acidity. The sulphuric plus salt was least toxic of all with a 
 pH of 6.3, as these larvae were still alive at the end of 23 days. The differ- 
 
 TABLE III. Resistance of whitefish larvae to acidity, neutrality, and alkalinity in 500 cc. 
 fresh boiled water and M/6 NaCl water; acid, acetic 0.529N sulphuric 0.485N, 
 hydrochloric 0.442N, COz gas added directly 
 
 O2 in 
 
 ee Chemical Used copnes pH Av. Time to Death Remarks 
 4 | .8 cc. acetic + boiled H,O 1.5 6.3 47 hrs. 
 41.8 CO ee ML OpNaGl ia eo Syke UE 
 4 18cc. ‘* + boiled H2O * FAO 25e0- 
 Ae AL SiCC eee NL OL Nal Lt 340 
 4 | .6 cc. HCl + boiled HO es 6.3 20s 
 ACTl..6) Coen t-te /6NaGl % ritstoy 
 4 | .26cc. “ + boiled H.O 4 7.0 isp 
 4 | .26cc. “* + M/6 NaCl % OTe es 
 4 | .6 cc. HeSO,4 + boiled HO 2 6.3 Cee ik 
 4 ees | PFE SE IM YAN ENG!| %E PAS) I alive 24 days 
 4 |.16cc. ‘“ -+ boiled H,O is 7.0 ds 
 4 6cc, ‘ + M/6 NaCl # cs 500 “ I alive 24 days 
 4 | control boiled water z 9.0 ger 
 4 le Oe Vi/GENaCl. beret See ie a: 87 ee 
 24 | CO2 + boiled water af 6.3 2 hrs. 25 min. Stock 3 
 45 “a ae ac Ai 6 zB 4 “ec “e 4 
 15 | boiled water i G.0 bites ea See: Saas 
 9 i‘ x Ai 9.0 Sees 
 15 | COs + boiled water 2.8 6.4 5 ‘ (not dead) we gl 
 18 ae os aé ae 2.8 6.4 12 “ec “ce “ec “ee 3 
 
 ence in the reactions to the three acids was small. The variation in the 
 hydrogen ions was probably influencing growth rather than the anions, sul- 
 phate, acetate, and chloride. This is in keeping with the work of Loeb (’04, 
 12) on the chemicals most influencing growth. He has studied the effect 
 of salt solutions on growth and regeneration and finds that the cations or 
 substituted hydrogen ions are most influential in changing growth, calcium, 
 sodium, and potassium being necessary at some time during the life cycle for 
 normal individuals to develop. 
 
 The effect of direct addition of carbon dioxide to water of high and low 
 oxygen was studied in relation to length of life in whitefish larvae. In low 
 oxygen the larvae died more rapidly in acid water (6.3) than in alkaline 
 
April, 1925 DEVELOPMENT OF THE WHITEFISH 1I2 
 
 (9.0). With high oxygen (3 cc. per liter) a pH of 6.3 did not kill, though 
 the exposure was for the greater part of two days. This is in accordance 
 with Well’s statement that large amounts of oxygen antagonize the detri- 
 mental effects of high concentrations of carbon dioxide. Statements of the 
 effect of the different combinations of oxygen and carbon dioxide in this 
 paper seem contradictory. The experiments were all started with different 
 stocks so the number of items that can be compared is unavoidably small. 
 However when the time to “ fin ray short”’ (series III, table II) is plotted 
 on cross section paper with oxygen and pH scales, and the points showing 
 equal time connected in a manner suggested by Huntington’s (’19) plots of 
 human death rate in relation to temperature and humidity, the results ap- 
 pear orderly (Fig. 2). The time for “ tail flat” in series II shows a similar 
 
 Oxygen in ce per liter 
 
 Fic. 2. Showing equal time lines on an oxygen-hydrogen-ion chart. The broken 
 line passes approximately through combinations of oxygen and hydrogen-ion concentra- 
 tion in which the embryos developed a flat tail in ten days, beginning four days after 
 spawning. The solid lines pass through combinations of oxygen and hydrogen-ion con- 
 centration in which the embryos developed short fin rays in 17 and 19 days respectively. 
 The general trend of these lines suggests that development may be expected to be most 
 rapid at about 3.4 c.c. of oxygen and pH 7.6 to 7.7, where the large cross is placed. This 
 cross is in the center of the ellipse. The center of the ellipse suggested by the flat tail 
 curve would fall at a higher H-ion concentration. 
 
 relation. Observations were not made often enough to bring out the neces- 
 sary details in any but series III, as noted above; accordingly this interpreta- 
 tion can be suggested only as a basis for further investigation. 
 
113 ADA R. HALL Ecology, Vol. VI, No. 2 
 
 Under experimental conditions eggs develop into normal embryos with a 
 pH of 7.0 and a much lower oxygen content than in the hatchery. Much 
 greater variations were produced in per cent hatching and in length of time 
 for development by varying the pH toward the acid or alkaline end of the 
 scale than by changing the oxygen. This is in accord with the idea of 
 Powers (’20). He concluded from his reconnaissance of the pH of Puget 
 Sound waters under varying conditions of tides, weather, etc., that pH has 
 more to do with compatibility of habitat than oxygen. 
 
 Effect of Conditions on Reactions of Fry from Different Stocks 
 
 As each of the stocks hatched, gradient experiments were run with the 
 larvae to see if the environment during the early embryonic life had any ef- 
 fect on the pH range which they would choose. A gradient tank suited to 
 the size of the larvae was used. This tank was 20.5 cm. by 2 cm. by 1.5. cm. 
 with screens 1.5 cm. from each end, and outlets at both sides of the middle. 
 Water was introduced drop by drop behind the screens at the ends, and 
 flowed out at the center. The end thirds were thus very nearly the same as 
 the water introduced there, while the center third was a mixture of the two. 
 
 ' Stock 1 and 2 were reared from the early germinal cap stage and the 
 elongation of the tail, respectively, in the vivarium waters. Both of these 
 stocks then lived through the period of heart and blood formation in the ex- 
 perimental waters. The results of the gradient experiments are suggestive. 
 No larvae hatched from the experimental dishes of the first stock, but those 
 hatching in the control (pH 7.8) stayed within the range of 7.6 to 8.2 when 
 given a choice of 6.6 to 9.0. Fig. 3 A shows the reactions of larvae of stock 
 2 to control water (7.8). When given a gradient of 6.4 to 8.2 a very definite 
 choice of end is seen. Larvae reared in water of moderate hydroxyl ion 
 concentration (pH 9.0, Fig. 2 B) choose the alkaline end of the tank, but 
 contrary to expectation those from the high hydrogen ion experiments (pH 
 6.4, C) also choose this end. None of the other larvae reared in any of the 
 later stocks or experiments choose this low hydrogen ion concentration when 
 there is water of a higher acidity available. 
 
 In stock 4 the eggs were raised to hatching at Put-in-Bay and so began 
 hatching a few hours after they were put into the experimental waters. 
 These therefore passed the time of heart and blood formation in the lake 
 water. Animals hatched on the 14th and 16th of March were kept in the 
 experimental waters, and gradients run with them on the 17th and 22d. 
 Nearly all of these showed the characteristic exploration of the whole tank 
 for from three to five minutes then a preference for the acid end of the tank. 
 The turning back occurred at a very definite pH and the avoidance was from 
 either end, for often when an animal entered the unfavorable water it stayed 
 there turning back from the changing point to again enter the unfavorable 
 
April, 1925 DEVELOPMENT OF THE WHITEFISH 114 
 
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 Fic. 3. Reaction of larvae of the four stocks to a permanent gradient. A long, 
 narrow tank was used with water of the desired pH entering at each end, making a 
 permanent gradient for the entire period of the experiment. The fish swam freely in 
 the tank for the time recorded in the marginal figures. A, control larva from stock 2. 
 B, larva from stock 2 raised through the period of heart and blood formation in a pH 
 of 8.6 to 9.0. C, larva from stock 2 raised through the period of heart and blood forma- 
 tion in a pH of 6.4. D, larva of stock 4 raised in lake water, but hatched in pH of 6.4. 
 E, larva of stock 4 hatched in a pH of 6.4 and given a choice of 8.0 io 9.0. 
 
115 ADA R. HALL Ecology, Vol. VI, No. 2 
 
 end several times before it escaped to the more favorable water. We there- 
 fore find the point of preference clearly marked. The fish hatched and 
 kept in water of a pH of 6.3 show a preference for the acid end of a gradient 
 (fig. 3 D). Those hatched and kept at 9.0 at first seemed to choose the 9.0 
 end of the gradient but they gradually worked down into the acid end and 
 stayed there. 
 
 Wells (15) states that if a gradient is entirely confined to pH above 
 neutrality, 8.0 to 9.0, that fish will choose the more alkaline end even though 
 preferring an acid pH when given a choice of 6.4 to 9.0. I found that this 
 was true of the whitefish larvae (fig. 3 EF). The larvae hatched in acid 
 water (pH 6.4) chose more exclusively the part from 8.8 to 9.0 than those 
 hatched in 9.0 water, although the latter turned at 8.4 and spent most of their 
 time in the 9.0 end. Yet both would normally choose the acid end of a gradi- 
 ent from 6.4 to 9.0. Wells believes that fish are avoiding pH 8.0, and indi- 
 cates that if given a choice of pH 8.0 and 9.0 they will choose the latter. 
 This seems to be a general reaction. Plankton studies of vertical distribu- 
 tion show the smallest number of individuals at the thermocline (pH 8.0) 
 with increasing numbers each side of it in either acid or alkaline waters. 
 
 The whitefish larvae reared through the period of heart and blood forma- 
 tion in experimental waters which were high in carbonate, and both quanti- 
 tatively and qualitatively different in salt content generally from the lake 
 waters from which the eggs were taken, behave in a gradient differently from 
 fish reared in lake water but hatched in experimental waters. This difference 
 lies in the direction of breaking up the preference, otherwise shown, for acid 
 rather than alkaline waters, causing all larvae to prefer a pH of 7.2 to 8.2 
 (regardless of the pH of the rearing waters). Gilbert (18) and Snyder 
 ((23) have shown that the salmon return to the tributary in which they were 
 hatched. These experiments suggest that the chemical condition of the water 
 during heart and blood formation modifies the reactions of fishes. The salt 
 content and the hydrogen ion concentration of streams up which salmon run 
 differ (Van Winkle ’14). These experiments suggest that the solution of 
 these migration problems may be comparatively simple. 
 
 Summary and Conclusions 
 
 1. Hydrogen ion concentrations favorable for fertilization are too high 
 for later development. Optimum hydrogen ion concentration is gradually 
 lowered as the embryo becomes older. 
 
 2. The most sensitive stages in whitefish development seem to be the 
 first cleavages and early gastrulation. 
 
 3. Fry hatching at one, two, and four months after spawning differ in 
 size of body but not in size of yolk; those hatching at four months are 4 to 
 6 mm. longer than those hatching earlier. The later fry also live longer than 
 those hatching earlier, in spite of having the same amount of yolk available. 
 
April, 1925 DEVELOPMENT OF THE WHITEFISH 116 
 
 4. Eggs raised through the period of heart and blood formation in water 
 high in carbonates and differing from their normal environment show a dif- 
 ferent type of gradient reaction to hydrogen ion concentrations. Such larvae 
 raised in both acid (6.3) and alkaline water (9.0) choose the alkaline end of 
 the gradient when 6.3 to 9.0 is offered. 
 
 5. When exposed to water with a pH of 6.3 obtained by adding CO, di- 
 rectly, the larvae died earlier in the acid water than in the alkaline with a low 
 oxygen content. A high oxygen content antagonizes the CO, present, pro- 
 longing the life of the larvae. 
 
 6. In a gradient of 8.0 to 9.0 larvae of both acid and alkaline hatching 
 environment choose the alkaline end (8.8 to 9.0). 
 
 I wish to thank Dr. V. E. Shelford, at whose suggestion this work was 
 undertaken, for his helpful advice and kindly criticism during the course of 
 the investigation. 
 
 The whitefish eggs were secured through the courtesy of the U. S. Bureau 
 of Fisheries from the hatchery at Put-in-Bay. I wish to thank Mr. Downing 
 and his associates for making my stay there both pleasant and profitable. 
 The Bureau of Fisheries extended me the privileges at Put-in-Bay through 
 Dr. H. B. Ward of the University of Illinois. 
 
 LITERATURE CITED 
 
 Clark, W. M. 1920. The determination of hydrogen ions. Baltimore. Williams and 
 Wilkins. 
 
 Clarke, F. W. 1920. The data of geochemistry. U. S. Geol. Survey Bull. 695. 
 
 Cohn, E. J. 1918. Studies in the physiology of spermatazoa. Biol. Bull, 34: 167-218. 
 
 Gilbert, C. H. 1918. Contributions to the life history of the sock-eye salmon (no. 5). 
 Rep. Comm. of Fisheries of British Columbia, 1918. 
 
 Hall, A. R. 1923. The effect of oxygen and carbon dioxide on the development of the 
 toad (Bufo americanus LeConte). Ecology, 5: 290-310. 
 
 Huntington, E. 1918. World power and evolution. Chapter V. New Haven. Yale 
 University Press. 
 
 Loeb, Jacques. 1904. On the influence of the reaction of the sea water on the regen- 
 eration and growth of Tubularians. Univ. of Cal. Pub. Physiol., 1: 139-147. 
 
 —. 1912. Mechanistic Conception of Life. Uniy. of Chicago Press. 
 
 Osterhout, W. J. V. 1914. Antagonism between acids and salts. Jour. Biol. Chem., 
 19: 517-520. 
 
 Powers, E. B. 10920.° The variation of the condition of sea water, especially the hydro- 
 gen ion concentration, and its relation to marine organisms. Pub. Pug. Sd. Biol. 
 Sta., 2: 369-385. 
 
 Shelford, V. E. 1918. Equipment for maintaining a flow of oxygen-free water and for 
 controlling gas content. Bull. Ill. State Lab. Nat. Hist., 11: 573-575. 
 
 Snyder, J. O. 1923. A second report on the return of salmon marked in 1915 in the 
 Kalamath River. Calif. Fish and Game, 9: I-90. 
 
 Wells, M. M. 10913. The resistance of fishes to different concentrations and combina- 
 tions of oxygen and carbon dioxide. Biol. Bull., 25: 323-347. 
 
 ——. 1915. Reactions and resistance of fishes in their natural environment to acidity, 
 alkalinity, and neutrality. Biol. Bull., 29: 221-237. 
 
 Van Winkle, W. 1914. The quality of the surface waters of Washington. U. S. 
 Geological Survey water supply paper No. 3309. 
 
VITA 
 
 Ada Roberta Hall was born in Florida in 1890. She received her 
 preparatory training in the Lincoln High School, Portland, Oregon, gradu- 
 ating in 1908. She taught in country grade schools until February, 1911, 
 when she entered the University of Oregon. During the period of her 
 undergraduate work she taught the primary grades at Fern Ridge and 
 Wendling, Oregon, for two years and a half, and was senior Playground 
 Director in the City of Portland Parks for three summers. She was labora- 
 tory assistant in Zoology at the University of Oregon during her senior year, 
 and was a member of Scroll and Script, Senior Honorary Society. She 
 received her A.B. degree with Highest Honors in Zoology in 1917. She 
 continued at the University of Oregon for two years as graduate assistant 
 in Zoology and received her A.M. degree iti 1919. The summer of 1918 was 
 spent at the Puget Sound Biological Station, Friday Harbor, Washington. 
 In the fall of 1919 she went to the University of Illinois as Fellow in Zo- 
 ology. During the year 1920-21 she was elected to Sigma Xi and in the fall 
 of 1920 to Iota Sigma Pi. The summer of 1920 was spent at the University 
 of Michigan Biological Station at Douglas Lake. She completed her work 
 for the degree of Doctor of Philosophy in June, 1921. During the period 
 of graduate.study she published the following papers: 1. Some Experiments 
 on the Resistance of Sea Urchin Eggs to Sulphurous Acid. Pub. Puget Sd. 
 Biol. Sta. 2:113-119. 2. Regeneration in the Annelid Nerve Cord. Jour. 
 Comp. Neur. vol. 33, No. 2:163-191. 
 
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