The British Journal of

Experimental Biology

THE OXYGEN REQUIREMENTS OF CERTAINAQUATIC ANIMALS AND ITS BEARINGUPON THE SOURCE OF FOOD SUPPLY.

BY W. J. DAKIN, D.SC., Professor of Zoology, University of Liverpool,and CATHERINE M. G. DAKIN, B.Sc.

CONTENTSSECTION A.

i. Experiments on the Oxygen Requirements of Goldfish .

Technique .Concluding Experiments

3. The Oxygen Consumption ofdeveloping Plaice Eggs .

Technique .

3023O4306

310

SECTION B.3. The Absorption of Oxygen in the

Course of Experiments . . 3 1 5

4. Summary 320

5. References 322IT is now about twenty years since Professor Putter of theUniversity of Bonn surprised most zoologists by assertingthat the main source of the food supply of aquatic animalsconsisted of organic matter in solution. His theory embracedthe aquatic animals of both fresh and marine waters.

It was natural that in the study of animal physiologya detailed investigation of animal nutrition should beginwith land animals. In fact our knowledge of this subjecthas almost been restricted to mammalia. Leaving asidespecial cases, it is characteristic of terrestrial animals thatthey capture prey in the form of other organisms ; they feedupon the vegetable products of the earth, or they live on amixed diet of animals and plants. Quite an extensive seriesof adaptations have been made out and there has been muchcorrelation of structure and function.

In that diverse and extensive assemblage of aquatic animalsVOL. 11.—NO. 3. 293 T

W. J. Dakin and Catherine M. G. Dakinranging from, say, the Coelenterata to the fishes one meetswith analogous (and homologous) structures apparently for theperception and capture of paniculate animal and plant food.Alimentary canals and related organs are present for itstransformation and absorption. It is not surprising, then,that the character of the food supply of most aquatic animalsshould have been (and still is) regarded as not unlike thatof terrestrial animals. It must, however, be admitted that itwas largely on the basis of analogies and not altogether onexperimental data that the ideas of twenty-five years agoregarding animal food supplies were founded. It was quitecommon to find that, although the structure of an alimentarycanal of some aquatic organism was known, there was littleinformation about the contents which might on occasion fillit. In whole collections of marine animals the alimentarycanals of the specimens were often quite devoid of contents.(This gap in our knowledge has been partially filled to-day.1Om)Supported by a few observations, the ultimate result of thedeductions drawn from the analogies alluded to above was thatbiologists and fishery experts came to regard the " plankton "as of profound importance in the food cycles of seas and lakes.The smallest floating plant-like organisms (unicellular plants-—diatoms and plant-like animals, flagellata, and so on) capturedwith tow-nets of bolting-silk were regarded as the link betweenthe dissolved mineral substances in the water and the materialsessential as animal food. The smaller animals of the planktonfed upon these microscopic organisms, to be devoured in turnby the larger planktonic animals, which again formed the foodof the fishes and even the whalebone whales. Many series ofthis type have been enumerated in works on plankton. Achain of this kind taken from Johnstone's Life in the Sea isthe following : " Peridians (plant-like organisms)—Copepoda—sprats—whiting—cod—man."8

With the inventions of Hensen dating from about 1885,the Kiel school of planktologists initiated their great effortto estimate quantitatively the actual amount of this floatinglife present in the sea at any time and throughout the year.Their efforts seemed to indicate that the coastal waters andinland seas at least were no barren water reservoirs. Thus

294

Oxygen and Aquatic Animalsin terms of dry organic substance, whilst cultivated land hasbeen calculated to produce 1790 kgs, per hectare, the un-cultivated Baltic Sea was shown to give 1500 kgs. per hectarein floating planktonic organisms. It is not surprising that theplanktologists did not doubt the sufficiency of the food suppliesof aquatic organisms if this was sought in the form of livingor dead animal and plant bodies.

Such was the position in 1907, when Putter published awork14 in which he claimed that actual experiment indicated :—

(1) that aquatic animals require more food to cover theirdaily requirements than is available in the formof paniculate food ("geformter Nahrung" in theoriginal—i.e. dead or living bodies of animals orplants, or particles of the same);

(2) that more organic food matter is actually availablein solution in a given volume of sea water than inthe form of particulate food in the same volume;

(3) that aquatic animals find their chief source of foodin the form of organic matter in solution.

Putter's first paper" was based upon chemical analysesand experimental work giving (a) the amount of carbonpresent in the form of organic compounds in solution in thesea water at Naples; (b) the amount of carbon required inthe form of food to cover the daily requirements of variousmarine animals; and (c) the amount of carbon available inthe plankton. Knowing (6) and (c) a simple arithmeticalcalculation would give the amount of sea water which thesupposed planktonic feeders .must necessarily filter in orderto obtain their daily requirements. The estimates of theamount of organic carbon present in solution in the seawater were obtained by direct analyses. The daily foodrequirements of various marine animals were estimated byfirst determining the oxygen requirements. The amount offood available in particulate form was calculated fromtabulations which had appeared in various papers from theKiel school of planktologists.

Obviously if the amount of organic carbon present insolution in sea water is normally very much in excess of

29s

W. J. Dakin and Catherine M. G. Dakinthat found in the form of paniculate food, there would beconsiderable reason for regarding the sea as a kind ofnutrient fluid. This conclusion would be confirmed if it couldbe shown that the plankton was only sufficient to meet thefood requirements, provided that the animals filtered extra-ordinary quantities of sea water per hour or day. Putterthought that he could prove both these statements to betrue. His first estimates were that one litre of sea watercontained 92 mgs. of carbon in solution, and that this wasabout 23,000 times as much as was present in the planktonicorganisms in the same volume !

Unfortunately for Putter, Henze6 was very soon able toshow that the analyses of the sea water were altogetherinaccurate, and that the 92 mgs. of carbon per litre had tobe reduced to something like 3 mgs.! This was confirmedby Raben of Kiel.19 As a matter of fact so far as mostanalyses available to-day are concerned, the amount of carbonin solution in the form of organic compounds in the open seafalls within the limits of experimental error.

One might easily assume that this discovery was quitesufficient to shatter Putter's theory altogether. However,although throwing doubt on some of his work, the chemicalinvestigations which followed seemed to show that the amountof food available in the form of planktonic organisms wasalso very small and equally insufficient to meet the foodrequirements which had been calculated from the oxygendeterminations. Some of Professor B. Moore's results gaveonly 0.4 mg. of C. per litre in the form of plankton. Theinterpretation of these results of physiological and chemicalinvestigation is by no means easy. Putter, in spite of somuch criticism, still adheres to his belief in the importance ofthe absorption of foodstuffs from solution.18 His estimatesmight have been more extensively tested, for only toofrequently one finds it assumed that this problem of nutritionis both quite simple and completely solved.

Investigations have already been made to obtain moreaccurate estimates of the particulate food available in bothsea and fresh water; the actual feeding habits of aquaticorganisms have been more closely followed by experiments.

296

Oxygen and Aquatic AnimalsBoth these lines of inquiry may be pursued further, especiallythe latter, in the light of Potts' work on Teredo}5 It is mostessential, however, that the food requirements, as calculatedby physiological experiment, should be subjected to revisionand analysed by further experiment. There is a wide fieldhere for investigation requiring the closest collaboration ofthe zoologist and the chemist.

The main feature of the work of Putter is bound up withthis last line of investigation and, whatever modifications thispart of his work may receive in the future, it has provedstimulating to research. His figures are to a large extentstill awaiting a refutation based on experiment from thosewho have most severely criticised his theory. Putter basedhis estimations of the amount of food required by aquaticanimals on the results of experiments to determine the oxygenconsumption during a period of one to twenty-four hours.In general, the animal was enclosed in a volume of water ina more or less elaborate container, and the amount of oxygenpresent was determined at the beginning and at the end ofthe experiment. Now the oxygen which has been consumedhas been utilised for the oxidation of the organic substancesof the animal body. If the oxidation were complete and weknew exactly the relative proportions of the substancesoxidised, it would be possible to calculate directly the amountof substance used up in twenty-four hours, and consequentlythe amount of food necessary to make good the loss. Inpractice, the results will only be approximate and shouldindeed give minimal figures. If proteid only were oxidisedone part would require 1.26 parts oxygen, one part carbo-hydrate in the same way would require 1.23 parts oxygen,and one part fat would require 2.88 parts oxygen. Theoxidation is incomplete, however, and we obtain only anapproximate measure of the actual metabolism.

The following table from Putter's work gives the resultsof some of these estimations, and it will be seen thatthe smaller organisms consume a relatively extraordinaryamount of oxygen per unit of dry weight, and that thereis apparently no direct relation between dry weight andmetabolism.

VOL. II.—NO. 3. 297 T2

W. J. Dakin and Catherine

Name of Animal.

SuberitesCucumariaCeriactis.RhizostomaCestusPterotracheaTethys .AequoreaMurexAplysia* .Galathea.PalaemonCalanus .Heliastes 18 c

,. 74 c.Scorpjena

>)Maenula .SaccharomyceMould .

s

Collozoum

Weight ofSpecimen

in gins.

6 0 01 5 02 0 0

232-073O570

152080-0

9-0

6-321-50-95000073O-2

13-21 8 0

570037-S

...

...O-I

Percentageof OrganicSubstance

(Dry).

7-616-49-53O-50-250-6i-3

abt. 0-415-01 5 01 5 015-015-016-220-12O-O2 0 02O-O

abt 15-0abt. 15-0

0-4

Temp,of

Experi-ment.

°C.

15-018-715-526-01 6 01 6 016-013-22 3 022-526-823-S17-723-722-13i-522-22 3 029-026Ol6-O

M. G. DakinOxygen Consumption

Perkg. and

Er. innigs.

I I - 2

i8-s32-022-O

3-9512-317-74-8

7 2 07 9 0

345-O518-0

3570-O6io-o358-0418-0IOO-O

464-0351300

116000-0IIIO-O

Perkg. of

OrganicSubstanceand perhr. inings.

148I ' 3335

4400158020501380f2OO48053O

23OO345O

238OO377OI77O2O8O

5OO232O

2360OO772O0O2770OO

PerAnimaland per

hr. inmgs.

0-670-2760-645-100-2880-702 6 9038s0-650-4947-4o-49C-OO26O-I224-77-5

64-017-4

*••O-III

Now physiologists had recognised before Putter's time thatmetabolism was not directly proportional to the weight or sizeof the animal, and as far back as 1883 Rubner suggested40 arelationship between surface area and basal metabolism. Hisview was further advanced by his later investigations and bythose of other physiologists, until practically it became elevatedinto a law—the so-called "body surface law." Putter insistedthat it was the active surface of the body, where oxygen andother exchanges took place, which really determined thebasal metabolism. He suggested, for example, that by takingthe lungs into account one might explain some of thediscrepancies in the comparison of the basal metabolism ofthe mammalia. It is singular how little reference has beenmade by physiologists to this possibility in recent years.Adopting this principle, Putter attempted to show that if themetabolism were correlated with the active surface area therewas a remarkable agreement between diverse organisms, andthat knowing this area in any particular example one might

298

Oxygen and Aquatic Animalslegitimately attempt calculations of food requirements. Acalculation of this kind which might be questioned isPutter's estimate of the food requirements of the Copepoda(Vergleichettde Physiologie, Gustav Fischer, Jena, 1911, p. 266).

Practically all the modern work on basal metabolism whichbears any approach to accuracy has been carried out onmammals. Benedict and Harris have shown2 that in oneand the same species—man—the so-called law of Rubneris only approximately valid and that various factors, such assex, age, and athletic training, affect the basal metabolism.Factors other than temperature influence the basal metabolism,viz., condition of the alimentary canal in regard to food andpsychic conditions.

It must be evident then that it is impossible to predictwith accuracy the metabolism and the food requirements ofa series of different animals by the application of the law ofbody surface or that of Putter's active surface. Recentchemical work shows indeed that animals are not all of thesame flesh, and that the food requirements of any speciesmust be calculated from experiments on that species. Besides,the actual area of the absorbing surface is only one factorin the efficiency of a respiratory organ. The composition ofthe membrane plays a great part, and there are other factorswhich make area a secondary feature. But the possibility ofaccuracy in theoretical calculations is more remote still, whenone realises the difficulty of recognising and of determiningthe active surface area in most cases.

We bring forward this criticism because we regard thefigures put forward by Putter for the food requirements ofcertain aquatic organisms as of peculiar interest. In fact itis this part of his work that most requires further investigation.Putter makes his actual experimental results agree with histheoretical calculations on the basis of the theory of surfacearea, and then draws on this agreement for support. Weconsider that any theoretical calculation of the food require-ments of an organism, made in this way, is interesting butaltogether unreliable, and to lump together all aquatic animalsas if their nutritional habits were likely to be the same is agrave error. We know of cases already, like the Corals,

W. J. Dakin and Catherine M. G. DakinConvoluta, and the White Ants, where peculiar symbioticconditions prevail. The future will probably show that wehave not realised the extent of symbiosis in regard to nutrition.There are likely to be numerous other causes of diversity.

We cannot dismiss Putter's figures, which are theactual results of chemical investigations as summarily ashis theoretical calculations. When, therefore, Putter calculates(in a criticism of a paper by W. J. Dakin *) that a Copepod(Ca/anus sp.) would have to catch and devour 9,750,000individuals of Thalassiosira nana daily in order to cover itsfood requirements, we are faced with a problem. The Copepodcould not obtain sufficient food in paniculate form if it hadto meet, these demands suggested by the results of the oxygendeterminations. But could it possibly meet such demandsby the absorption of food in solution ? We suggest thatthese high oxygen figures require further explanation.

It is impossible to obtain any real idea of the foodrequirements of an animal by a few experiments at one season,and grave errors readily creep into calculations based on theresults of experiments with five or six specimens.

The food requirements of a crab which were calculated fromexperiments made on one or two individuals at the breedingseason could not be safely applied to the whole year. Forexample, Johnstone has shown9 how the constitution of variousmarine fishes varies regularly throughout the year, and inparticular how in the herring there are " seasonal metabolicphases." These are of the two categories (1) "those whichmake up the annual reproductive cycle, and (2) those whichare to be related particularly to the annual wave of seatemperature." These features are probably common to manyother aquatic animals. This being the case, it would be verydangerous to compare even accurate estimations of the foodrequirements of a species when metabolism was at its highestwith the food available in the sea water at another season andperhaps at some other place. This seems a legitimate criticismto bring forward against Putter's oxygen determinations.We believe, however, that much of this experimental workmust be repeated. It is only too easy for aquarium experimentsto give abnormal results, and the interpretation of normal

300

Oxygen and Aquatic Animalsresults is not always easy. The oxygen consumption of agoldfish an hour after a meal is much higher than twelve hoursafterwards. Which of these figures approximates most closelyto the average in nature? If, throughout the year, the foodrequirements of a number of aquatic organisms are as highas the oxygen determinations would seem to indicate, thereis a factor present which still remains unknown.*

We may now turn to the experimental side, and to thecalculations of Putter based upon actual feeding and starvationexperiments which led to our own work with goldfish, axolotls,and plaice eggs. The most elaborate series of investigationsof this kind were those made on fish.16 One of these willserve as a type of the others, and the species is that whichwe have also used.

A small number of goldfish were divided into three groups.Group I. was used for analysis. Group II. lived without anyfood in tap water renewed daily (later on a small amount ofvarious salts was added). Group III. were given organicmatter in solution—asparagine and glycerine. The goldfishwhich received no food, Group II., lived forty-two days,whilst those of Group III. lived up to fifty-six and seventy-eight days. Furthermore, the oxygen analyses indicated thatthe Group II. fishes diminished by 39 per cent, of theirweight during the experiment. The Group III. fishes notonly lived much longer, but were using 20 per cent, moreoxygen than the Group II. examples at the end of theexperiment; they had not lost so much weight, owingapparently to the presence of asparagine and glycerine.

Putter assumed that the artificial solutions did not containall the necessary substances required as food, but that therecould be no question about the utilisation of dissolved carboncompounds. He also added (Zeit. /. allgem. physiologie, Bd.ix., p. 222): "There is no doubt that a nutrition without

* It should be pointed out that estimates of the paniculate food available(organisms or detritus) in the sea or fresh water are probably also minimal figures.Lohmann showed long ago that the plankton nets failed to capture the smallerorganisms and the recent work of Allen' has emphasised this. And of course acomplete calculation of the food so available during a whole year at any particularplace could only be used in connection with animals which do not migrate, followingup their food supplies. We are not specially concerned with this aspect of thequestion in the present paper.

301

W. J. Dakin and Catherine M. G. Dakindissolved foodstuffs is possible, and it is not impossible thatcases of this kind are realised in nature. But the experimentsat Naples show that the fish in the Naples aquarium underapproximately natural conditions, obtain one-half to three-quarters or more of their food requirements by the absorptionof dissolved food."

Now in any case the actual duration of life of the fishin Plitter's goldfish experiments must not be taken into undueconsideration. We have kept goldfish of approximately thesame length and size alive for periods varying from a fewweeks up to three months when all food, particulate or insolution, has been excluded. The duration of life dependsupon the condition of the fish at the beginning of theexperiment. Putter did not employ enough specimens tocut out individual variation and a chance result. It willalso be seen that the oxygen consumption may diminish by60 to 75 per cent, before the animals die.

After we had commenced our experiments we found thatLipschiitzn had repeated some of Putter's experiments withfish, and that his conclusions were not favourable to Putter.This position seemed only to strengthen the necessity forfurther investigation, especially since we had ourselvesexperienced rather varied results at the beginning when wewere using only a few specimens.

SECTION A.

1. Experiments on the Oxygen Requirements of Goldfish.

We commenced our experiments with goldfish by keepinghalf a dozen in glass jars holding several litres of water. Twofish were fed chiefly with so-called ants' eggs (pupae); the fishin the other jars were given no solid food, but were, in onecase, left without anything and in another were given thesame substances which Putter used—asparagine and glycerine,and in the same strength. The water in the jars was emptiedout and renewed morning and evening to prevent the growthof any organisms, and every morning the jars were cleanedwith hydrochloric acid. It was found that the addition ofa small quantity of NaCl to the water was of advantage.

302

Oxygen and Aquatic AnimalsChemically pure salt was used. No oxygen determinationswere made in the first experiment at all. The purpose wasto find out if there were an appreciable difference betweenthe length of life of fish kept in tap water and those in tapwater with added organic compounds, and in particular tocut sections of the tissues from the dead fish in order to seewhat changes, if any, had taken place. The first fish whichdied was one of the couple kept in ordinary tap water. Ithad lived without food for fifty-four days. The other fishin this jar lived, however, as long as the two in the waterto which asparagine and glycerine had been added. Aftersixty-five days (it was the beginning of the vacation) all threewere killed and sectioned. No difference whatever could bemade out between the tissues of the fish from the pure waterjar and those from the jar containing asparagine and glycerine.There was a marked difference, however, between the tissuesof all these fish and those of the specimens which had beenfed on solid food. The fish not fed with solid food wereundoubtedly starved. The most marked difference wasobvious in the loss of fat and in the condition of the musclefibres. In sections these were clearly seen to have becomeattenuated, whilst an increase of the surrounding connectivetissue had apparently taken place. In reality this was onlyrelative. Almost exactly the same histological condition wasobserved in a goldfish which Professor Johnstone (Ocean-ography Department, University of Liverpool) examined aboutthis time. It bore a large sarcoma which had either preyedon the other tissues or had resulted in the fish starving,for the muscle fibres quite remote from the cancer regionwere greatly attenuated, whilst the connective tissue sur-rounding them was relatively conspicuous. It may be statedthat the fish from the experimental jars were outwardly inperfect condition when they were .killed, if judged solely onthe appearance of their scales and their fins. And this is agood test of their condition. They were, however, inactiveand rested on the bottom for long periods. They had alsolost weight. We realised from this experiment that morethan six specimens would be required for a definite resultand that there seemed, so far as we had gone, to be absolutely

3°3

W. J. Dakin and Catherine M. G. Dakinno difference between fish in water with organic compoundsand those in water alone.

The results of our first oxygen determinations served as awarning against carrying out physiological work of this kindon a small scale. We had been lucky in our first venture toescape any outbreak of disease. For several months followingthis we were so troubled that we encountered almost all thepossible goldfish diseases, the worst being Chilodoniasis dueto a Ciliate (Chilodon cyprini) and Gyrodactyliasis caused bythe trematode genus Gyrodactylus.

It was thought at first that possibly the handling andthe experimental conditions, whilst the oxygen determinationswere being made, was responsible for the outbreaks. Anexperiment with starved fish that were not used for oxygendeterminations showed that this deduction was wrong. Theintense development of parasitic disease commenced after thefish had been without particulate food for two or three weeks,and it did not matter whether asparagine and glycerine hadbeen present on not. On the other hand the fish fed with ants'eggs flourished no matter how much they were handled. Someof these fish which had been discarded and removed to thelaboratory preparation room were in water which was oftenleft unchanged for days and was at times quite murky. Theysurvived. Nothing could have been more certain than theindication that withdrawal of solid food left the fish open toan intense attack of the parasites which had unfortunatelyinfected our stock. Months elapsed before we had a safesupply again, and in the meantime we had developed thatpeculiar knowledge of the conditions favourable for the animalswhich can only come after long practical experience. We werealso able to spot a fish which was unwell at an early stage.

At this point we were rather in favour of the view thatthe goldfish in water with asparagine and glycerine consumedmore oxygen than the others and really did utilise the dis-solved organic matter. It cannot be said, therefore, that wewere experimenting with set views against Putter's theory.

Technique.—The methods adopted for the later experimentswere as follows : The goldfish were kept separately in litreglass cylinders. These were arranged in two series and all the

3°4

Oxygen and Aquatic Animalsgoldfish were paired—that is to say, whenever one that hadbeen given food in solution was tested for the oxygen con-sumption, its fellow of the pair which had been in plain waterwas also examined. A great effort was made to choosegoldfish of the same size, and indeed to choose those withapproximately the same oxygen consumption. The jars wereall well aerated with a special pump made for us by theAerograph Co., London. Oxygen determinations were madeby placing the fish in large stoppered bottles (3000 c.c.) whichwere kept under water in a great slate tank which had acapacity of 41.5 gallons. In this way we had a means ofkeeping the temperature constant for all the bottles duringa long experiment. The oxygen content of the water wasestimated by the Winkler method before the experimentcommenced, by collecting with an indiarubber siphon threeor four samples from the jars in the tank. For greateraccuracy we increased the size of the samples for the Winklertest until they were 365 c.c. After taking off three or foursamples in this way, the fish to be used were carefully placedinto the submerged bottles which were then closed, thestoppers being inserted under water. At the end of varyingperiods of two, three, six hours, etc., the bottles were takenout of the large slate tank and opened carefully on a table. Asiphon was rapidly inserted and a quantity of water drawnoff for the oxygen test. The difference between the oxygencontents of the bottle before and after the experiment repre-sented the amount used by the fish.*

It is necessary to state that we found the simple methodjust described quite as accurate as the more complicatedmethods which have been tested. Several details are, however,of importance in this respect. The experiments were onlyof such short duration (three hours in the best series) as toensure that only a small proportion of the total oxygen availablehad been consumed by the fish, and only a small quantity ofwaste matter added to the water. Numerous controls of all kindsconvinced us that these results approached a normal more closelythan experiments of twenty-four hours' duration without changeof water, but where efforts had been made to supply oxygen.

• It is quite unnecessary to detail the chemical methods involved in Winkler'sprocess for the quantitative determination of dissolved oxygen in water.

305

W. J. Dakin and Catherine M. G. DakinIn any case it must not be forgotten that the figures are

not used as absolute. In no case has an oxygen determinationbeen made on a fish from water containing asparagine andglycerine without its corresponding plain tap water controlgoing through exactly the same procedure. The whole seriesof experiments have been comparative, and any errors affectingone side have also affected the other.

The Concluding Experiments.—Twenty-four goldfish weretaken from a large supply, kept under observation until theyappeared free from parasites and in good condition. Twenty-four jars were arranged quite arbitrarily in two rows of twelveon a long bench. Then to cut out any personal choice, thelaboratory attendant "tossed" a coin to decide which seriesshould receive glycerine and asparagine and which seriesordinary water. The fish were labelled A-L and A'-L', andA and A' formed a pair, B and B' another pair, and so on.Six fish on each side were utilised for oxygen determinations,and the other six fish on each side left as further controls onthose which were being more frequently handled.

The first oxygen determinations were made after all thefish had been starved for two days, an important point,because we found that the oxygen consumption of fish thatwere being well fed varied very considerably. Absence offood for forty-eight hours brought down the oxygen con-sumption quite considerably. Here again is a point worthnoting. There is more likelihood of the oxygen require-ments of the first few days of starvation being a normalrequirement than that of well - fed laboratory specimens.The same probably applies to other experiments of thisnature. What evidence is there that under natural conditionsthe animals employed in experiments ever capture as muchfood as they have been consuming in the aquaria ?

Following the first oxygen determinations made in thiscase on 12th October, the tests were repeated on variousdates until 5th December, a period of fifty-four days (com-pared with the forty-two days of Putter's experiment).Fortunately the first deaths from starvation occurred amongstthe controls which were not being used for the oxygen deter-minations, and so we were able to run our comparisons fora fairly long time. The incidence of the deaths was, how-

306

Oxygen and Aquatic Animalsever, rather interesting, and showed how easily misconceptionsmay arise when working with small numbers or over a shortperiod. Four fish died from the series in plain tap waterbefore one died from the series in water with glycerineand asparagine. We should undoubtedly have taken thisas a proof that the fish in the water with the reagentsmentioned were utilising these substances in some way, hadit not been that the oxygen determinations lent no supportto this. Even so we were altogether puzzled, and it wasnot until deaths occurred in the same condition in the otherseries that we began to realise that this distribution wasprobably nothing but coincidence, and was due to originalvariations amongst the fish. The termination of theexperiment was almost a "dead heat," for on 19th Januaryone fish was left in each series and these two died withinfour days of each other—more than three months after theexperiment was commenced! There was no evidence fromthis result that the presence of asparagine and glycerine hadsupplied any essential quantity of food. It was, however,the oxygen determinations upon which we mainly relied.The results of these (leaving out working calculations andgiving the milligrammes of oxygen consumed per hour at acommon temperature of 15° C.) are shown in the followingtables. The corrections for temperature were made byapplying the equation Q10 = 2.5. This was obtained fromoxygen determinations made at different temperatures betweenio° and 15° C.

Tables I., II., III., and IV. and the corresponding curvesFigs. 1, 2, and 3 show quite clearly that no difference can bedistinguished between the oxygen consumption of the fish inordinary tap water and those in the water to which glycerineand asparagine had been added.

The series A, B, C and A', B', C (Fig. 1) are the bestfor comparison because from the first the duration of theexperiment was three hours, the period which was found laterto be most satisfactory. The series D, E, F and D', E', F '(Fig. 2) were one-hour experiments, and it was noted thatmore variation occurred in the results. These variationsdisappear somewhat if the total oxygen consumption of thefish at each'date is compared (Fig. 3). Eventually the series

3°7

W. J. Dakin and Catherine M. G. Dakin

TABLE I.

Goldfish in Tap Water with Glycerin* and Asparagine. Results calculated for 15° C.

Date.

Fish.A

B

C

Dec 12.

1-917

I-I55

i-6o8

Oct 15.

1-280

0-859

I - I I I

Oct 18.

0-989

0-92

1-30

Oct. 22.

o-9M

0-84

I-2O

Oct 39.

0803

072

1-14

Nov. 1.

0-756

0-52

0-856

Nov. 12.

O495

0-452

O-773

Nov. 23.

0-550

0-302

0-613

Dec 5.

0-487

0-46

O-43'

r Mg. ofOxygen used

-1 per hourduring

I Experiment.

TABLE II.Goldfish in Plain Tap Wattr. Results calculated for 150 C.

Date.

Fish.A'B'

C

Dec. 12.

1-548

1-894

1-597

Oct 15.

0-881

1-29

O-955

Oct. 18.

1-158

1-43

0-989

Oct 22.

1-359

1-18

1-04

Oct 29.

1030

102

072

Nov. 1.

O596

072

0-589

Nov. 12.

0-642

0-62

0-420

Nov. 23.

0-477

0-49

0-551

Dec 5.

0-461

0-42

0-450

r Mg. ofOxygen used

per hourduring

. Experiment.

TABLE III.

Goldfish in Tap Water with Glycerin* and Asparagine. Results calculated for 15° C.

Date.

Fish.D

EF

Total

Oct 15.

1-49

172

2-03

5-24

Oct. 18.

1-40

i-i3

i-38

4-91

Oct. 32.

145

1-15

i-43

4-03

Oct 30.

o-?9

I-2O

'•34

3-43

Nov. 14.

109

0865

0-728

2-683

Nov. 19.

0-68

0-63

...

c Mg. ofOxygen used

per hourduring

. Experiment

...

TABLE IV.

Goldfish in Plain Tap Water. Results calculated for 15* C.

Date.

Fish.D'

E'

F

Total

Oct 15.

1-301

0-989

1-131

3-421

Oct 18.

0-965

0-810

1-29

3-O74

Oct 22.

O-8I5

0-758

1-28

2-853

Oct 30.

108

0-443

I-I2

3-643

Nov. 14.

0-863

0-763

0-728

3-354

Nov. 19.

0-50

O53

. . .

Mg. ofOxygen used

per hourduring

. Experiment.

...

308

Oxygen and Aquatic Animals

Ott.tr ir a t

FlC. I.—Oxygen consumption of the fish A, B, and C in tap water + asparagine andglycerine ; and A', B', and C in ordinary tap water.

Oiis

FIG. a.—Oxygen consumption of the fish D, E, and F in tap water + asparagine andglycerine ; and D7, E', and F' in ordinary tap water.

VOL. II.—NO. 3. 3°9 u

W. J. Dakin and Catherine M. G. DakinD, E, F, etc., was not tested as frequently as the series A, B,C, etc., but it was used for other experiments.

It will be remembered that Putter found that his goldfishin water with organic compounds lived over i£ times as longas those in ordinary tap water and consumed i | times the

Octlff 19 23 SO No*. 19FIG. 3.—Total oxygen consumption of the fish D, E, and F, and D', E', and F'.

amount of oxygen. There is no indication of any such effectin our experiments, which are in agreement with those ofLipschiitz.11

2. The Oxygen Consumption of developing Plaice Eggs andits Relation to the Amount calculated from Analyses ofthe Composition of Plaice Eggs at Different Stages" ofDevelopment.

It should be clear from our discussion of Putter's theorythat we regard that author's calculations of the food ofcertain aquatic animals from their oxygen consumption, andfrom analyses, as the section of greatest importance andmost in need of further investigation. As part of thatinquiry Putter made several experiments to show that theoxygen consumption of certain aquatic animals deprived ofsolid food was greater than could be accounted for on theloss of dry weight which took place. In other words,absorption of food in solution must have taken place, althoughit might not have been sufficient to prevent starvation. Thusin the goldfish experiments of Putter the oxygen determina-tions gave 832 mgs. as the oxygen consumption, whilst only365-375 mgs. oxygen were really necessary for the oxidation

310

Oxygen and Aquatic Animalsof the substances consumed, according to calculations fromthe loss of weight which had occurred.

Unfortunately in all the experiments of this kind made upto date one has had to analyse certain animals at the beginningof the experiment, and that was naturally the end of them.The final analyses were made on a different set of specimenswhich had been utilised for the experiment. It was necessary,therefore, for approximate accuracy that the animals shouldhave all been of the same composition at the beginning ofthe experiment. This can seldom be realised in practice, forthe wet weight (if it can be obtained) is not a reliable guideto dry weight or composition, and the same criticism mightbe raised against other theoretical methods of computation.

We endeavoured, therefore, to take advantage of theopportunity presented by a fish hatchery at the BiologicalStation of Port Erin to make a test on plaice eggs. Thesecould with a little trouble be counted and, if thousands wereused (of the same age), any individual variation might beconsidered as eliminated. One could take a sample of anyage from a hatching box of eggs and analyse them. Followingthis the oxygen consumption of the eggs of the same boxcould be determined every day, and after several days anotherbatch taken for analysis. If the embryos depended solely onthe food stores within the egg membranes, the oxygen con-sumption during the period of the experiment should agreewith that calculated from any loss of weight which mightoccur in the substance of the eggs.

Technique.—Two samples of eggs were taken from ahatching box which was supposed to contain eggs of aboutthe same age. Most were about the 8- or 16-cell stage. Theywere measured out (with as little sea water as possible) bymeans of an ordinary measuring glass, previous tests havingshown that one could approximate to within a few hundredin this way, and that it required about 2500 eggs to give awet weight of roughly 10 gms., an amount deemed necessaryfor accuracy in analysis. The eggs were then turned outon to an ample supply of clean filter paper and carefully driedof sea water by rolling them over and over very gently. Atthis stage they were counted, and to obviate any delay beforeweighing several helpers took a hand so that only a few

3 "

W. J. Dakin and Catherine M. G. Dakinminutes were involved. Each sample was next placed in aweighed Soxhlet thimble and weighed accurately on a chemicalbalance. This gave the wet weight. The thimbles were nextdried at about 85° C. for the dry weight. In the meantimethe oxygen consumption was being determined, using othereggs from the same hatching box.* These oxygen deter-minations were repeated day after day and always on samplesfrom the same box until fourteen days had elapsed. Twomore samples of eggs were then taken for analysis and countedand weighed in the same way as the first samples. The countsand analyses are as follows:—

Experiment commenced 1 \th April:—

Sample of Eggs A.Wet weight .Dry substanceWater .FatResidue

Nitrogen in 0.3 gm. Residue

Sample of Eggs AA.Wet weight .Dry substanceWater .Fat .Residue

Nitrogen in 0.3 gm. Residue

Experiment ended i$th April:—

Sample of Eggs B.Wet weight .Dry substanceWater .Fat .Residue

Nitrogen in 0.3 gm. ResidueSample of Eggs BB.

Wet weight .Dry substanceWater .Fat .Residue

Nitrogen in 0.3 gm. Residue

No. of eggs—2982.10.240 gms.0.721 „9-5*9 ..0.009 it0.71a 110.0436 „

No. of eggs—2945.9.676 gms.0.701 „8.965 „0.008 „0.694 „0.0429 „

No. of eggs—2470.8.11 T gms.

• 0.529 ..

• 7-58* ,,0.034 „0.497 „0.0413 „

No. of eggs—2250.7.549 gms.

• 0.458 „

• 7-091 .»0.015 ..0.444 „0.0431 „

• Numerous determinations of oxygen consumption of plaice eggs had previouslybeen made so that the experimental errors were reasonably well known, and suchdetails as the number to take and the best duration of experiment had beendiscovered. (Details will be published in another paper.)

3 "

Oxygen and Aquatic AnimalsSince we are dealing with large numbers of similar eggs

these results may be reduced to a common denominator,namely, the composition of 2000 eggs, and we may take theaverage of the two samples.

Composition of 2000 eggs at beginning and end of experiment,of experiment:—

2000 eggs.

Wet weight .Dry substance

At beginning

6.720.479

gms.

Water .Fat .Residue

Nitrogen in 0.3 gm. ResidueProteid calculated from Nitrogen

At end of experiment:—2000 eggs,

Wet weight .Dry substance

Water .Fat .Residue

Nitrogen in 0.3 gm. ResidueProteid calculated from Nitrogen

6.2510.0057

°-47330.0432

°-4»59

6.638 gms.

6.22030.0204

O-39730.04220.3476

From these figures it may be estimated that 2000eggs have lost 78.3 mgs. of proteid during fourteen days'development. Now the oxygen determinations which weremade during the fourteen days of the experiment show that2000 eggs in the hatching boxes used approximately somethingbetween 82.086 and 98.0 mgs. of oxygen. Assuming that thiswas used entirely in the oxidation of proteids, it correspondsroughly to a loss of between 65.67 and 77.8 mgs. of the latter,a figure which does not exceed (does not reach) the amountof proteid actually lost.

The interpretation of the result along the lines of Putteris that the developing fish eggs have not imbibed substancesin solution in the sea water, their own contained stores havesufficed.

The consumption of oxygen during development ascalculated from the loss of organic matter in the eggs duringthe experiment balances roughly the actual consumption asdirectly measured, that is to say, it is not found to be

VOL. II.—NO. 3. 313 U2

W. J. Dakin and Catherine M. G. Dakinonly half or a third of the latter (as in the case of Putter'sexperiments on other animals where food in solution wassupposed to be absorbed). The method of experiment seems,therefore, to be sound, but the result is only an approxi-mation.

Where an animal starved of paniculate food is absorbingany significant quantity of food by way of solution, this typeof experiment would indicate the fact. Where, however, onlya few per cent, of the total supply comes from solution itwould not be safe. Nor would it be safe to say that a smalldiscrepancy between, the actual oxygen determination and thetheoretically calculated amount from analyses indicated anabsorption of food from solution. The oxygen consumptionmay be accurately determined by actual experiment, but thefood requirements suggested by the amount must be regardedas approximate only. In the case of the plaice eggs, forexample, a very unexpected result has been found, for thereis three times as much fat at the end of the period ofdevelopment as at the beginning. The total amounts, 6 and20 mgms. are, however, so small that we must not stress thisresult. This fat must have come from the proteids present,for there is no evidence so far that plaice eggs contain anycarbohydrates to speak of. But as yet we have no evidenceto show what complex reactions have taken place in theseeggs. The discovery apart from our present investigationis an interesting one. The fact is that the metabolism ofthe invertebrata and lower chordata is a vast field that isnot to be covered with one general and simple formula.Aquatic organisms are not to be dumped together in a class,as if the mere fact that they are aquatic compels them tofunction in the same manner. The problems of human andmammalian metabolism are indeed complicated enough. Thereis no reason to believe that the different groups of the loweranimals present conditions which are less complex. Thus,F. A. Potts l t in an interesting paper on Teredo has shownthat this aquatic mollusc feeds upon wood and may die ifthe wood is deficient in amount. It has only very recentlybeen shownta that the white ants are unable to feed onwood—their apparent normal diet—unless large numbers ofcertain protozoa live within their alimentary canals. There

Oxygen and Aquatic Animalsare probably numerous other cases of a similar nature inaddition to those now recognised (like Convoluta, etc.)as symbiotic. Again few physiologists and extremely fewzoologists seem to realise that the elasmobranch fishes andthe teleost fishes are physiologically far apart. They appearto have much in common, yet the blood of the former hasthe freezing point of the external medium and varies withit, whilst that of the latter is more or less constant.6

It would surely be rash to calculate the food requirementsof an elasmobranch from those of a bony fish, grantinga knowledge of the gill areas in the two cases. Thereis no point in adding further examples. Where a foodrequirement is calculated for an aquatic organism, actualoxygen determinations and analyses of its composition mustbe carried out, and the metabolism and seasonal habits of theparticular organisms must be thoroughly well studied beforefar-reaching deductions are made.

SECTION B.

3. The Absorption of Oxygen in the Course of Experiments madeto determine the Oxygen Consumption of Certain AquaticAnimals.

Several investigators who have attempted the determina-tions of oxygen consumption of aquatic animals have remarkedupon a difference between the rate during the successive hoursof an experiment. The matter is an important one, and it isnecessary to know something of the cause and amount ofthis variation in order to interpret the results. It would bevery easy to overestimate oxygen requirements if the con-sumption during twenty-four hours were taken to be twenty-four times that of the first hour of experiment, and thishappened to be abnormally high. An extreme example ofthe kind to which we refer is the following from theinvestigations of the late Professor B. Moore. A spongeused 1.43 mg. of oxygen in eight and a half hours, but only1.57 in forty-eight hours. The crab Cancer used 17.79 mgs.in four hours, and only 19.49 mgs- when the experiment wascontinued up to nineteen hours. (Other results of Mooreare, however, too varied altogether to illustrate the point.)

31S

W.#J. Dakin and Catherine M. G. DakinBrunow8 found that the oxygen consumed by the crayfishAstacus fluviatilis diminished as his experiment continued.Thus in one case for sixty minutes the rate of oxygen con-sumption per hour and animal was 0.786 mg., whilst duringa 120-minutes experiment the rate fell to 0.667 mg-> a n d fora 3-hour experiment the oxygen consumption was only0.506 mg. per animal per hour. The cause of this veryconsiderable reduction was not worked out. In some experi-ments, however, in which the crayfish were out of water, theproduction of COS seemed to exert a retarding influence onoxygen consumption. Lipschtltzu found the same kind ofretardation in his work with goldfish. The following figuresare from his paper :—

Duration of experiment- 59 minutes ( O x ^ e n c ° n s u m P t i o n 1 O.82o mg.I. per animal per hour )

» i> 37^ „ ,, ,, 0.626 ,,

59 ., •> ., °-645 .»350 » .. ,. o-593 „

60 „ „ „ 1.45340 ,, ,, „ 1.03

58 ,, ,, » 1-33 „

344 >i .. 11 0-854 ,,

Lipschutz was indeed rather concerned about the errorwhich might arise by computing the oxygen consumption intwenty-four hours from experiments of short duration. Butit must be noted in this connection that he regarded theamount used in the first hour of experiment as too high,and due to the increased excitement of the fish owing tohandling. It is a probable theory, but was not proved byexperiment.

There are several possible ways of accounting for thediminution in the oxygen consumption per hour (if it actuallydoes occur) during a prolonged experiment. They are asfollows: (1) Handling the specimens and so causing anabnormally high rate of consumption at the commencementof the experiment. (2) The gradual absorption of oxygen

316

Oxygen and Aquatic Animalsand the consequent diminution in the amount available inthe closed vessel resulting in retarded oxygen consumptionduring the latter part of an experiment. (3) Accumulationof COS or of other waste compounds acting as a retardinginfluence on oxygen consumption and metabolism during thelatter part of the experiment. (4) The animals may have fedimmediately preceding the experiment.

All these are interesting possibilities with importantbearings upon the experiments made in connection withPutter's theory, whilst Nos. 2, 3, and 4 are of widerinterest.

So far as our knowledge at present extends, the inverte-brate groups do not present any unity in their reactions tochanges in the partial presence of oxygen. Mammalia arenot affected by pure oxygen at 760 mm. barometric pressure,whilst this is apparently deadly for the leech. Konopacki1(>

has shown that the oxygen consumption of the earthwormvaries with the pressure of the oxygen, and may be calculatedfrom the formula

a = kj~d

where a = oxygen consumption, k = a constant, and d = thepartial pressure of the oxygen. The same thing apparentlyholds good for Litnax.

On the other hand, Henze found that whilst the oxygenconsumption of Actinia and Anemonia varies in this waywith the reduction of the normal oxygen content of the water,other invertebrates are very independent. Amongst thesewere Echinoderms, certain pelagic molluscs, Crustacea {Car-cinus mcenas and Scyllarus latus), the molluscs Aplysia andEledone, and certain fishes.

In order to test the matter we first carried out experimentson plaice eggs. These were taken because they seemedexcellent examples on which to rule out the action of handling.The eggs were constantly in motion in the hatching boxes andtheir insertion into the experimental bottles did not evennecessitate removing them from this water. They weresimply run in with the water and the counts made at theend of the experiments. There could not be any psychicinfluences here!

317

W. J. Dakin and Catherine M. G. DakinThe results of several experiments are as follows :—

Experiment 1.—1000 eggs use 0.071 mg. oxygen in 1 hour„ „ 0.113 „ ,, 2 hours

Available oxygen reduced by 4.6 per cent in first hour.Experiment 2.—1000 eggs use 1.052 mg. oxygen in 6 hours

o.838„ 0.647

„ „ 0.366>, >• ° - * 3 6

Experiment 3.—1000 eggs use 0.088» o-io5

Experiment 4.— rooo eggs use 0.1200.210

5421 hour1

2 hours

The results are in remarkable agreement in that the oxygenconsumption of plaice eggs in a closed volume of sea water(the amount of oxygen available was considerable) was alwaysgreater during the first half-hour or hour than during thesecond and third. The effect of handling is ruled out here,and consequently we regard the results as indicating in thecase of plaice eggs a retardation of oxygen absorption dueto reduction of the amount of oxygen available, or to theincrease in the water of COt and excreted substances, or ofboth. Since the total amount of oxygen available in thevolume of water containing the eggs has been reduced by avery small percentage, the eggs are either very susceptibleto oxygen pressure, or else it is the second possibility referredto above which is the most important factor; and this maybe accentuated by the relatively more quiescent condition ofeggs during the experiment, although the jars were frequentlyturned over.

Similar experiments were made using the goldfish, axolotls,and the freshwater mollusc Anodon. A goldfish (specimen C)was placed for two hours in a very small jar of water, capacity808 c.c, containing only 7.97 mgs. of oxygen available insolution. It used up oxygen at the rate of 0.3804 mg. perhour. The same fish was then tested in a large jar containing7050 c.c. of water with 69.20 mgs. of oxygen available insolution. The amount of oxygen consumed was practicallythe same, viz., 0.3884 mg. per hour. The experiment wasrepeated with another fish, and again the difference was withinthe experimental error and the usual amount of variation.

318

Oxygen and Aquatic AnimalsIn this case the fish actually used more oxygen per hour inthe jar containing only 7.9 mgs. available oxygen than in thevessel containing 70.4 mgs. of available oxygen.

More extensive experiments were made with two axolotls,varying the duration of the experiment from two to twenty-fourhours. No appreciable reduction was noticed in the amountof oxygen consumed per hour. If any diminution took place,it was less than the experimental error. Finally, we examinedtwo Anodons which had been under observation for sometime. The following are the results for mussel A :—

Date.

Feb. 19 .

M

i

) 2°, 22, 28ar. 4, 5, 6, 71 : l

, 12. 13

Temp.

I2-O12-5I2-O13-512-513-013-513-513-016-016-0

Duration ofExperiment.

1 hour18 hours22 ,5 >5 ,5 .55 >5 >S .

, 10 mms.. 15 .,

Oxygen availablein Experimental

Vesselat beginning.

Mg.3I-29528-2732-16245-361*i6-27t327615-53+26-0732-2913-65+22-Ojt

Oxygen usedper hour.

Mg.O-78370-93030-87610-762O-3540-7950-6090-8430-8150-3050-865

* More oxygen available because large jar used Actually less oxygen per c.ct Oxygen contents reduced by addition to tap water of a certain amount of tap water raised tc-

boiling-point and then cooled.

In the case of both mussels an increase in the durationof the experiment from one hour to eighteen hours did not causeany appreciable diminution in the rate of oxygen consumption.It will be noticed that in this experiment the reduction in theamount of oxygen available at the end of eighteen hours wasroughly 50 per cent. Where the amount of available oxygenwas reduced by this amount at the beginning of the experimentby the addition of non-aerated water a very appreciabledifference in the oxygen consumption was apparent. Thelast experimental test of 13th March shows, however, that areduction by about 33 per cent, does not make a big difference.We may conclude, therefore, that the axolotls, the goldfish, andthe freshwater mussels are all somewhat independent of theoxygen contents of the water under normal conditions, andthat it is only when the amount of oxygen available falls

319

W. J. Dakin and Catherine M. G. Dakinbelow a certain minimum that oxygen consumption isprofoundly affected. We should say also that handling didnot produce any effect in the experiments quoted above,because the specimens were so accustomed to the treatmentreceived.

Moore's results can be explained upon these lines, in thatthe amount of oxygen available was too small for the durationof his experiments and fell below the minimum.

It is quite possible that handling was the cause of thevariation in oxygen consumption in Lipschiitz's fish experi-ments, but his suggestion that the greater variation whenlarge specimens were used supported this view is not altogethersatisfactory. Larger specimens would also use more oxygenand produce more COj. We intend carrying out furtherexperiments on the fish eggs in order to test their sensitivityto CO,

4. Summary.

1. The original theory of the food supply of aquaticanimals put forward by Putter, and based upon the resultsof certain experiments and analyses, claimed that the chiefsource of food of such animals was organic matter dissolvedin the sea, in lakes, rivers, etc., and that this was absorbeddirectly and indeed often by the gills, if present. Theposition he took up may be emphasised by his statementregarding fishes : " There is no doubt that a nutrition withoutdissolved foodstuffs is possible, and it is not impossible thatcases of this kind are realised in nature. But the experi-ments at Naples show that the fish in the Naples aquariumunder approximately natural conditions obtain one-half to three-quarters or more of their food requirements by the absorptionof dissolved food."

2. It is quite possible that small quantities of organicmatter in solution in water are absorbed by aquatic animals,and in some cases (particularly amongst protozoa living underspecial conditions) this may be an important, perhaps themost important, source of food. It is also possible that verysmall quantities of organic matter in solution may eventuallybe found to exercise a very profound influence (acting likevitamines, for example) on the life of aquatic animals.

320

Oxygen and Aquatic AnimalsEvidence for such is not evidence for the main thesis set upby Putter on the results of his own experiments.

3. The food requirements of many aquatic animals ascalculated by Putter on the basis of oxygen consumption areoften remarkably high and need further investigation. It isquite possible that there is an unknown factor at work here.In connection with these calculations we consider that purelytheoretical computations based upon the measurement orestimation of the active surface area of the body are notpermissible, and that the application of the law of surfacearea in connection with metabolism must not be allowed tosupplant experiment

4. Experiments on goldfish similar to those made byPutter, show that specimens kept in tap water without anyparticulate food live for varying periods (which are often ofconsiderable duration) dependent upon the original conditionof the fish, and the freedom of the experimental tanks fromparasites. The addition of the organic compounds, glycerineand asparagine, makes no difference to the duration of life,and the consumption of oxygen by the fish living in tapwater with these compounds does not exceed that of thecontrol fish in tap water only. Gradual starvation takesplace, and sections show that the mass of muscle tissuebecomes gradually reduced.

5. The cessation of feeding on particulate food makesthe fish particularly susceptible to the attacks of parasites[Chilodon cyprini and Gyrodactylus, sp.), if there is any chanceof such infection.

6. The consumption of oxygen by plaice eggs duringtheir development agrees fairly well with the amount computedfrom analyses of the composition of young eggs and eggsshortly before hatching, but the results are only approximate,although they fit in with the assumption that such floatingeggs have their own food stores and absorb nothing fromthe sea water.

7. Aquatic organisms are not to be grouped in one classin so far as nutrition and metabolism are concerned.

8. It had frequently been noted that when the oxygenconsumption of aquatic animals is measured, and the deter-

3 "

W. J. Dakin and Catherine M. G. Dakinmination extends over several hours there is a gradual fallingoff during the experiment. It is necessary to look for thisin every case before estimating the normal oxygen con-sumption over long periods. The variation may be due tohandling the specimens at the beginning of the experiment,to the gradual reduction of the oxygen available, to theaccumulation of waste products, or to time of feeding.

We have found that goldfish, axolotls, and Anodon usedin our experiments are to a certain extent independent of theoxygen pressure, which may fall considerably (until a certainminimum is reached) before the oxygen consumption of theanimals per hour is affected.

5. References.1 Allen (1919), Journ. Marine Biol. Assoc. of U.K., New Series, 12, No. 1.1 Benedict and Harris (1919), Biome trie Study of Basal Metabolism in Man, Carnegie

Institute of Washington.8 Brunow (1910), "Der Hungerstoffwechsel der Flusskrebses," ZeiLf. allg. physiol.,

12.*» Cleveland (1934), Biol. Bull., 48.4 Dakin, W. J. (1908), " Food of the Copepoda," Internal. Revue d. Hydrobiologie, 1.6 Dakin, W. J. (1912), "Aquatic Animals and their Environment," Internat. Revue

d. Hydrobiologie, p. 54.« Henze (1909), Pfluger's Archiv.f. d. ges. Physiol., 128.7 Henze (1910), "Ober d. Einfluss des Saurestoffdruckes auf d. gaswechsel einiger

Meerestiere," Biochem. Zeit., 2ft8 Johnstone, J. (1908), Life in the Sea, Cambridge University Press.0 Johnstone, J. (1918), Dietetic Value of the Herring, Reports Lanes, and Western

Sea Fisheries Committee, 1917, Liverpool.10 Konopacki (1907), "Ober d. Atmungsprozess d. Regenwurmen," Bull, de PAcad.

des Sciences de Cracovie.1Oa Lebour, Marie V. (1919), Journ. Marine Biol. Assoc. of U.K., New Series, 12;

1923, 13, No. 1.11 Lipschiitz, A., "Zur Frage iiber die Ernahrung der Fische," Zeit.f. allg.physiol., 12.18 Moore, Edie, e tc (1912), " Nutrition of Marine Animals," Biochem. Journ., 6.13 Potts, F. A. (1923), "Structure and Function of Liver of Teredo," Proc. Cambridge

Phil. Soc. Biol. Sc, 1.14 Putter, A. (1907), "Die Ernahrung der Wassertiere," Zeit.f. allg.physiol.u Putter, A. (1909), "Die Ernahrung der Wassertiere," Jena,G. Fischer.18 Putter, A. (1909), "Die Ernahrung der Fische," Zeit.f. allg.physiol., 9.17 "Studien d. vergleich. PhysioL des StoffwechseL" Abhand. d. Jkgi. Gesell. d.

wisstn. t. Gottingen. Math. Physiol. Klasse. Neue Folge, 6.u Putter, A. (1923), Biol. Centralbl^ 42, Nr. 2.10 Raben (1910), Wissensch. Meeresuntersuch., Kiel u. Helgoland, Neue Folge, 11,

Leipzig.80 Rubner (1883), Zeit.f. Biol., lft

322